Compositions and Methods Comprising Prostate Stem Cell Antigen (PSCA) Chimeric Antigen Receptors (CARs)

ABSTRACT

The present disclosure provides modified immune cells or precursors thereof (e.g. T cells) comprising a chimeric antigen receptor (CAR) capable of binding human PSCA. CARs capable of binding human PSCA, and nucleic acids encoding the same are also provided. Provided herein are bispecific CARs capable of binding human PSCA and human PSMA, nucleic acids encoding the same, and modified immune cells comprising the same. Modified immune cells comprising a PSMA CAR and a PSCA CAR are also provided. Compositions and methods of treatment are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 17/017,238, filed Sep. 10, 2020, which is entitled to priority under35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/985,808filed Mar. 5, 2020, and U.S. Provisional Patent Application No.62/898,896 filed Sep. 11, 2019, which are hereby incorporated byreference in their entirety herein.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in XML format and is herein incorporated by reference in itsentirety. Said XML copy, created on Sep. 7, 2023, is named046483_7280US2_SequenceListing.xml and is 246,233 bytes in size.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common cancer diagnosis and the secondleading cause of cancer-related death in American men. Despite recentadvances in the detection and treatment of localized disease,significant challenges remain in the management of this disease. Currentdiagnostic modalities are limited by a lack of specificity and aninability to predict which patients are at risk to develop metastaticdisease. Prostate-specific antigen (PSA) is effective at identifying menwho may have prostate cancer but is often elevated in men with benignprostatic hyperplasia, prostatitis, and other nonmalignant disorders.PSA and other current markers fail to discriminate accurately betweenindolent and aggressive cancers. There is no effective treatment for the20-40% of patients who develop recurrent disease after surgery orradiation therapy or for those who have metastatic disease at the timeof diagnosis. Although hormone ablation therapy can palliate thesepatients, the majority inevitably progress to develop incurableandrogen-independent disease.

There is a need in the art for compositions and methods for treatingprostate cancer. The present invention addresses this need.

SUMMARY OF THE INVENTION

The present invention is based on the finding that prostate stem cellantigen (PSCA) chimeric antigen receptor (CAR) T cells exhibit potentanti-tumor activity. The present invention is also based on the findingthat bispecific CARs and dual CARs capable of binding PSCA and prostatespecific membrane antigen (PSMA) exhibit significantly enhancedanti-tumor activity.

Accordingly, in certain aspects, the instant disclosure provides achimeric antigen receptor (CAR) comprising an antigen binding domaincapable of binding prostate stem cell antigen (PSCA), a transmembranedomain, and an intracellular domain.

In certain exemplary embodiments, the CAR is capable of binding prostatestem cell antigen (PSCA), and comprises an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21, 23, 25, or 27.

In another aspect, the instant disclosure provides a bispecific chimericantigen receptor (CAR) comprising an extracellular domain comprising anantigen-binding domain capable of binding prostate stem cell antigen(PSCA), and an antigen-binding domain capable of binding prostatespecific membrane antigen (PSMA), a transmembrane domain, and anintracellular domain.

In certain exemplary embodiments, the CAR is capable of binding prostatestem cell antigen (PSCA) and prostate specific membrane antigen (PSMA),and comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 40, 42, 80, 81, or 82.

In another aspect, the instant disclosure provides a nucleic acidcomprising a polynucleotide sequence encoding a CAR, wherein the CAR iscapable of binding prostate stem cell antigen (PSCA), and comprises anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 21, 23, 25, or 27.

In certain exemplary embodiments, the CAR is capable of binding prostatestem cell antigen (PSCA), and is encoded by a polynucleotide sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQID NO: 20, 22, 24, or 26.

In another aspect, the instant disclosure provides a nucleic acidcomprising a polynucleotide sequence encoding a bispecific CAR, whereinthe CAR is capable of binding prostate stem cell antigen (PSCA) andprostate specific membrane antigen (PSMA), and comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 40, 42, 80, 81, or 82.

In certain exemplary embodiments, the bispecific CAR is encoded by apolynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 39 or 41 or 79.

In another aspect, the instant disclosure provides a vector comprising anucleic acid comprising a polynucleotide sequence encoding a CAR,wherein the CAR is capable of binding prostate stem cell antigen (PSCA),and is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20, 22, 24, or 26.

In another aspect, the instant disclosure provides a vector comprising anucleic acid comprising a polynucleotide sequence encoding a bispecificCAR, wherein the CAR is capable of binding prostate stem cell antigen(PSCA) and prostate specific membrane antigen (PSMA), and wherein thebispecific CAR is encoded by a polynucleotide sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39 or41 or 79.

In certain exemplary embodiments, the vector is an expression vector.

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof comprising a CAR, wherein the CAR iscapable of binding prostate stem cell antigen (PSCA), and comprises anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 21, 23, 25, or 27.

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof, comprising a bispecific CAR, wherein thebispecific CAR is capable of binding prostate stem cell antigen (PSCA)and prostate specific membrane antigen (PSMA), and comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 40, 42, 80, 81, or 82.

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof comprising a vector that comprises anucleic acid comprising a polynucleotide sequence encoding a CAR,wherein the CAR is capable of binding prostate stem cell antigen (PSCA),and is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20, 22, 24, or 26.

In certain exemplary embodiments, the modified immune cell or precursorcell thereof further comprises a PSMA-CAR, wherein the PSMA-CARcomprises an antigen-binding domain, a transmembrane domain, and anintracellular domain.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a modifiedimmune cell or precursor cell thereof comprising a CAR, wherein the CARis capable of binding prostate stem cell antigen (PSCA), and comprisesan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 21, 23, 25, or 27.

In another aspect, the instant disclosure provides a method of treatinga disease in a subject in need thereof, comprising administering to thesubject an effective amount of a modified cell or precursor cell thereofcomprising a CAR, wherein the CAR is capable of binding prostate stemcell antigen (PSCA), and comprises an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21,23, 25, or 27.

In certain exemplary embodiments, the disease is a cancer.

In certain exemplary embodiments, the disease is prostate cancer.

In certain exemplary embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIG. 1 illustrates the generation of a prostate stem cell antigen(PSCA)-specific CAR. A PSCA CAR comprised of an scFv from a humanizedanti-PSCA Ab (2B3) was constructed and cloned into retroviral vectorMSGV.

FIG. 2 illustrates CAR expression of T cells electroporated with invitro transcribed RNA of a PSCA CAR with 4-1BB and CD3z domains(2B3.BBZ) and/or a PSMA CAR with 4-1BB and CD3z domains (J591.BBZ)(upper panel), and PD-L1-Fc staining of a PSCA CAR co-electroporatedwith bispecific antibody 10A5-1412 (an aPDL1-aCD28 bispecific Ab;indicated as 10A5) or TGFB3-1412 (an aTGFbRII-aCD28 bispecific Ab;indicated as TGFB3).

FIG. 3 illustrates CD107a expression of RNA co-electroporated T cellsstimulated with PC3-PSCA-PSMA or K562. T cells were electroporated witha PSCA CAR with 4-1BB and CD3z domains (2B3.BBZ) and/or a PSMA CAR with4-1BB and CD3z domains (J591.BBZ), as indicated.

FIG. 4 illustrates T cells lentivirally transduced with a PSCA (2B3) CARwith a mutated ICOS signaling domain (ICOS.YMNM) showed improved lyticcapability, decreased cytokine production in vitro and equivalent invivo antitumor activities as a 4-1BB signaling PSCA CAR. PSCA CARs witheither ICOS or ICOS.YMNM signaling domain were constructed and clonedinto a lentiviral vector. The CAR expression levels were comparable witheither 4-1BB or CD28 signaling domain CARs (upper). CAR-T cells werestimulated with PSCA positive cell lines PC3.PSCA.PSMA.CBG orPC3.PSCA.PSMA.CBG.PD-L1 and examined for cytokine (IL-2 and IFN-gamma)production (lower panel). Human lung cancer cell line A549 and humanbreast cancer cell line MDA468 were also used. Abbreviations: PSCA (2B3)CAR with ICOS and CD3z domains (2B3.ICOSz or PSCA.2B3.ICOSz); PSCA (2B3)CAR with variant ICOS (YMNM) and CD3z domains (2B3.ICOSz.YMNM orPSCA.2B3.ICOSz.YMNM); PSCA (2B3) CAR with CD28 and CD3z domains (2B3.28zor PSCA.2B3.28z); PSCA (2B3) CAR with ICOS and CD3z domain co-expressedwith a PD1-CD28 switch receptor (PD1.282B3.ICOSz orPD1.28.PSCA.2B3.ICOSz); PSCA (2B3) CAR with variant ICOS (YMNM) and CD3zdomain co-expressed with a PD1-CD28 switch receptor(PD1.282B3.ICOSz.YMNM or PD1.28.PSCA.2B3.ICOSz.YMNM); PSCA (2B3) CARwith 4-1BB and CD3z domains (2B3.BBz or PSCA.2B3.BBz); PSCA (2B3) CARwith 4-1BB and CD3z domains (2B3.BBz) co-expressed with a PD1-CD28switch receptor (PD1.28.2B3.BBz or PD1.28.PSCA.2B3.BBz); andnon-transduced (NO TD or NTD).

FIG. 5A illustrates results from a CD107a assay of T cells expressingPSCA CARs with either an ICOS or ICOS.YMNM signaling domain.Abbreviations used in FIG. 5A are the same as those in FIG. 4 .

FIG. 5B illustrates results from a killing assay for T cells expressingPSCA CARs with either an ICOS or ICOS.YMNM signaling domain.Abbreviations used in FIG. 5B are the same as those in FIG. 4 .

FIG. 6 illustrates results from experiments using a PC3-PSCA-CBG-PDL1tumor model. For lentiviral transduced (LVV TD) T cells, 1e⁶ cells permouse were i.v. (intravenous) injected at day 21 days post tumorinoculation. 5 mice were tested in each group. Abbreviations used inFIG. 7 are the same as those in FIG. 4 .

FIG. 7 illustrates the average radiance of tumors after subcutaneousinjection with various PSCA CARs. Abbreviations used in FIG. 7 are thesame as those in FIG. 4 .

FIG. 8 illustrates tumor sizes after subcutaneous injection with variousPSCA CARs. Abbreviations used in FIG. 8 are the same as those in FIG. 4.

FIG. 9A is a schematic of the bi-specific CAR vectors used for studiesencoding the PSMA (with 2F5 scFv) and PSCA (with 2B3 scFv)-targetedCARs, linked with a Gly4Ser element. Abbreviations used in FIGS. 9A-9D:non-transduced (NTD); PSMA (2F5) CAR with 4-1BB and CD3z domains(2F5-BBZ); PSCA (2B3) CAR with 4-1BB and CD3z domains (2B3-BBZ); PSMA(2F5) and PSCA (2B3) bispecific CAR with 4-1BB and CD3z domains(2F5-S-2B3-BBZ; Gly4Ser linker); PSMA (2F5) and PSCA (2B3) bispecificCAR with 4-1BB and CD3z domains (2F5-S-2B3-BBZ; (Gly4Ser)4 linker); PSCA(2B3) and PSMA (2F5) bispecific CAR with 4-1BB and CD3z domains(2B3-S-2F5-BBZ; Gly4Ser linker); PSCA (2B3) and PSMA (2F5) bispecificCAR with 4-1BB and CD3z domains (2B3-L-2F5-BBZ; (Gly4Ser)4 linker).

FIG. 9B illustrates surface expression of the CARs on lentivirustransduced CAR T cells at the end of the primary expansion.

FIG. 9C illustrates the percentage of lentivirus transduced CAR T cellsthat express the PSMA or PSCA-CAR, or PSMA-PSCA bispecific CAR stainingwith human recombinant PSMA-Fc and PSCA-His protein, as measured throughflow cytometry.

FIG. 9D illustrates CAR T cells comprising the indicated CAR wereco-cultured with targets for 4 hours and the percentage CD107aexpression was quantified on CD8 positive cells.

FIG. 10A illustrates results from CAR T cells co-cultured with PC3-PSCAcells (Effector:Target ratio=1:1). Supernatants were obtained 24 hoursafter co-culture, and cytokine production was analyzed by ELISA.Abbreviations used in FIGS. 10A-10D are the same as used in FIGS. 9A-9D.

FIG. 10B illustrates results from T cells tested for their cytolyticactivity at indicated E:T ratios for 8 hours against PC3-PSCA cells.

FIG. 10C illustrates results from CAR T cells co-cultured with PC3-PSMAcells (Effector:Target ratio=1:1). Supernatants were obtained 24 hoursafter co-culture, and cytokine production was analyzed by ELISA.

FIG. 10D illustrates results from T cells tested for their cytolyticactivity at indicated E:T ratios for 8 hours against PC3-PSMA cells.

FIGS. 11A-11B illustrate the finding that TGFbR-IL12R switch receptorscan boost T cell function. FIG. 11A, upper right panel, shows IFN-gammaproduction of NK cells trasferred with TGFbR-IL12R co-cultured withK562, with or without TGFb1 in the cultures. FIG. 11A, lower rightpanel, shows pSmad staining of T cells transferred with TGFbR-IL12Rswitch receptors after stimulation with TGF beta. FIG. 11B showscytokine production of NY-ESO-1 positive tumors stimulated NY-ESO-1 TCRtransduced T cells, co-transferred with TGFbR-IL12R switch receptors.

DETAILED DESCRIPTION

The present invention provides compositions and methods for modifiedimmune cells or precursors thereof (e.g., modified T cells) comprisingchimeric antigen receptors (CARs) capable of binding prostate stem cellantigen (PSCA). In certain embodiments, the invention providescompositions and methods for modified immune cells or precursors thereofcomprising bispecific CARs (e.g PSCA & PSMA), PSCA CARs with a dominantnegative receptor (e.g., TGFbRDN), PSCA CARs with a switch receptor(e.g., PD1/CD28 or TGFbR/IL12R), and PSCA CARs in combination withbispecific antibodies (e.g., PD-L1/CD28). The provided compositions andmethods are useful for treating cancer (e.g. prostate cancer).

It is to be understood that the methods described in this disclosure arenot limited to particular methods and experimental conditions disclosedherein as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements, Molecular Cloning: A Laboratory Manual(Fourth Edition) by MR Green and J. Sambrook and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (2013, 2nd edition).

A. Definitions

Unless otherwise defined, scientific and technical terms used hereinhave the meanings that are commonly understood by those of ordinaryskill in the art. In the event of any latent ambiguity, definitionsprovided herein take precedent over any dictionary or extrinsicdefinition. Unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein is well-knownand commonly used in the art. The methods and techniques provided hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

That the disclosure may be more readily understood, select terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including virtually all proteins or peptides, can serveas an antigen.

Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to elicit the desired immune response. Moreover,a skilled artisan will understand that an antigen need not be encoded bya “gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “downregulation” as used herein refers to the decrease orelimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited toan amount that when administered to a mammal, causes a detectable levelof immune suppression or tolerance compared to the immune responsedetected in the absence of the composition of the invention. The immuneresponse can be readily assessed by a plethora of art-recognizedmethods. The skilled artisan would understand that the amount of thecomposition administered herein varies and can be readily determinedbased on a number of factors such as the disease or condition beingtreated, the age and health and physical condition of the mammal beingtreated, the severity of the disease, the particular compound beingadministered, and the like.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly about 10 amino acids and/or sugars in size.Preferably, the epitope is about 4-18 amino acids, more preferably about5-16 amino acids, and even more most preferably 6-14 amino acids, morepreferably about 7-12, and most preferably about 8-10 amino acids. Oneskilled in the art understands that generally the overallthree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity andtherefore distinguishes one epitope from another. Based on the presentdisclosure, a peptide used in the present invention can be an epitope.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anarginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

The term “immunosuppressive” is used herein to refer to reducing overallimmune response.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides. Itwill be understood that when a nucleotide sequence is represented by aDNA sequence (i.e., A, T, C, G), this also includes an RNA sequence(i.e., A, U, C, G) in which “U” replaces “T.”

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

A “target site” or “target sequence” refers to a nucleic acid sequencethat defines a portion of a nucleic acid to which a binding molecule mayspecifically bind under conditions sufficient for binding to occur. Insome embodiments, a target sequence refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (α) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver. A transplant can alsorefer to any material that is to be administered to a host. For example,a transplant can refer to a nucleic acid or a protein.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to, Sendaiviral vectors, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

B. Chimeric Antigen Receptors

The present invention provides chimeric antigen receptors (CARs) capableof binding prostate stem cell antigen (PSCA). In certain embodiments, asubject CAR comprises an antigen binding domain capable of binding PSCA,a transmembrane domain, and an intracellular domain. In another aspect,the invention includes a bispecific CAR comprising an extracellulardomain comprising an antigen-binding domain capable of binding prostatestem cell antigen (PSCA) and an antigen-binding domain capable ofbinding prostate specific membrane antigen (PSMA), a transmembranedomain, and an intracellular domain.

Also provided are compositions and methods for modified immune cells orprecursors thereof, e.g., modified T cells, comprising the CAR. Thus, insome embodiments, the immune cell has been genetically modified toexpress the CAR. Nucleic acids encoding said CARs, vectors encoding saidnucleic acids, and modified cells (e.g. modified T cells) comprisingsaid CARs, vectors, or nucleic acids, are also provided.

A subject CAR of the invention comprises an antigen binding domaincapable of binding PSCA, a transmembrane domain, and an intracellulardomain. A subject CAR of the invention may optionally comprise a hingedomain. Accordingly, a subject CAR of the invention comprises an antigenbinding domain capable of binding PSCA, a hinge domain, a transmembranedomain, and an intracellular domain. A subject bispecific CAR of theinvention comprises an extracellular domain comprising anantigen-binding domain capable of binding prostate stem cell antigen(PSCA) and an antigen-binding domain capable of binding prostatespecific membrane antigen (PSMA). In embodiments where the CAR is abispecific CAR, the antigen-binding domain capable of binding PSCA islinked to the antigen-binding domain capable of binding PSMA. In someembodiments, the PSCA antigen-binding domain may be N-terminal to thePSMA antigen-binding domain. In some embodiments, the PSMAantigen-binding domain may be N-terminal the PSCA antigen-bindingdomain. The C-terminal antigen binding domain of a bispecific CAR of theinvention may be operably linked to another domain of the CAR asdescribed herein.

The antigen binding domain may be operably linked to another domain ofthe CAR, such as the transmembrane domain or the intracellular domain,both described elsewhere herein, for expression in the cell. In oneembodiment, a first nucleic acid sequence encoding the antigen bindingdomain is operably linked to a second nucleic acid encoding atransmembrane domain, and further operably linked to a third a nucleicacid sequence encoding an intracellular domain.

The antigen binding domains described herein can be combined with any ofthe transmembrane domains described herein, any of the intracellulardomains or cytoplasmic domains described herein, or any of the otherdomains described herein that may be included in a CAR of the presentinvention. A subject CAR of the present invention may also include ahinge domain as described herein. A subject CAR of the present inventionmay also include a spacer domain as described herein. In someembodiments, each of the antigen binding domain, transmembrane domain,and intracellular domain is separated by a linker.

Antigen Binding Domain

The antigen binding domain of a CAR is an extracellular region of theCAR for binding to a specific target antigen including proteins,carbohydrates, and glycolipids. A subject CAR of the invention comprisesan antigen binding domain capable of binding prostate stem cell antigen(PSCA).

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. In someembodiments, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof. The choice of antigen binding domain maydepend upon the type and number of antigens that are present on thesurface of a target cell.

In some embodiments, the antigen binding domain is selected from thegroup consisting of an antibody, an antigen binding fragment (Fab), anda single-chain variable fragment (scFv). In some embodiments, a PSCAbinding domain of the present invention is selected from the groupconsisting of a PSCA-specific antibody, a PSCA-specific Fab, and aPSCA-specific scFv. In one embodiment, a PSCA binding domain is aPSCA-specific antibody. In one embodiment, a PSCA binding domain is aPSCA-specific Fab. In one embodiment, a PSCA binding domain is aPSCA-specific scFv.

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) of an immunoglobulin (e.g., mouse or human) covalentlylinked to form a VH::VL heterodimer. The heavy (VH) and light chains(VL) are either joined directly or joined by a peptide-encoding linker,which connects the N-terminus of the VH with the C-terminus of the VL,or the C-terminus of the VH with the N-terminus of the VL. In someembodiments, the antigen binding domain (e.g., PSCA binding domain)comprises an scFv having the configuration from N-terminus toC-terminus, VH-linker-VL. In some embodiments, the antigen bindingdomain comprises an scFv having the configuration from N-terminus toC-terminus, VL-linker-VH. Those of skill in the art would be able toselect the appropriate configuration for use in the present invention.

The linker is usually rich in glycine for flexibility, as well as serineor threonine for solubility. The linker can link the heavy chainvariable region and the light chain variable region of the extracellularantigen-binding domain. Non-limiting examples of linkers are disclosedin Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014/087010,the contents of which are hereby incorporated by reference in theirentireties. Various linker sequences are known in the art, including,without limitation, glycine serine (GS) linkers such as (GS)_(n),(GSGGS)_(n) (SEQ ID NO:119), (GGGS)_(n) (SEQ ID NO:120), and (GGGGS)_(n)(SEQ ID NO:121), where n represents an integer of at least 1. Exemplarylinker sequences can comprise amino acid sequences including, withoutlimitation, GGSG (SEQ ID NO:122), GGSGG (SEQ ID NO:123), GSGSG (SEQ IDNO:124), GSGGG (SEQ ID NO:125), GGGSG (SEQ ID NO:126), GSSSG (SEQ IDNO:127), GGGGS (SEQ ID NO:128), GGGGSGGGGSGGGGS (SEQ ID NO:129) and thelike. Those of skill in the art would be able to select the appropriatelinker sequence for use in the present invention. In one embodiment, anantigen binding domain of the present invention comprises a heavy chainvariable region (VH) and a light chain variable region (VL), wherein theVH and VL is separated by the linker sequence having the amino acidsequence GGGGSGGGGSGGGGS (SEQ ID NO:129), which may be encoded by thenucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQID NO:130).

Despite removal of the constant regions and the introduction of alinker, scFv proteins retain the specificity of the originalimmunoglobulin. Single chain Fv polypeptide antibodies can be expressedfrom a nucleic acid comprising VH- and VL-encoding sequences asdescribed by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883,1988). See, also, U.S. Pat. Nos. 5,132,405 and 4,956,778; and U.S.Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvshaving inhibitory activity have been described (see, e.g., Zhao et al.,Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63;Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al.,Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have beendescribed (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7;Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit RevImmunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66).

As used herein, “Fab” refers to a fragment of an antibody structure thatbinds to an antigen but is monovalent and does not have a Fc portion,for example, an antibody digested by the enzyme papain yields two Fabfragments and an Fc fragment (e.g., a heavy (H) chain constant region;Fc region that does not bind to an antigen).

As used herein, “F(ab′)2” refers to an antibody fragment generated bypepsin digestion of whole IgG antibodies, wherein this fragment has twoantigen binding (ab′) (bivalent) regions, wherein each (ab′) regioncomprises two separate amino acid chains, a part of a H chain and alight (L) chain linked by an S—S bond for binding an antigen and wherethe remaining H chain portions are linked together. A “F(ab′)2” fragmentcan be split into two individual Fab′ fragments.

In some embodiments, the antigen binding domain may be derived from thesame species in which the CAR will ultimately be used. For example, foruse in humans, the antigen binding domain of the CAR may comprise ahuman antibody or a fragment thereof. In some embodiments, the antigenbinding domain may be derived from a different species in which the CARwill ultimately be used. For example, for use in humans, the antigenbinding domain of the CAR may comprise a murine antibody or a fragmentthereof.

In certain embodiments, the antigen binding domain capable of bindingprostate stem cell antigen (PSCA) comprises a heavy chain variableregion that comprises three heavy chain complementarity determiningregions (HCDRs). HCDR1 comprises the amino acid sequence superfamly (SEQID NO: 1), HCDR2 comprises the amino acid sequence WIDPENGDTEFVPKFQG(SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence TGGF (SEQ IDNO: 3). The antigen binding domain capable of binding prostate stem cellantigen (PSCA) also comprises a light chain variable region thatcomprises three light chain complementarity determining regions (LCDRs).In certain embodiments, LCDR1 comprises the amino acid sequenceSASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acid sequenceDTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequenceQQWSSSPFT (SEQ ID NO: 6).

In certain embodiments, the heavy chain variable region (VH) of theantigen binding domain capable of binding prostate stem cell antigen(PSCA) comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 and/or the lightchain variable region (VH) comprises an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

In certain embodiments, the antigen binding domain capable of bindingprostate stem cell antigen (PSCA) is selected from the group consistingof a full length antibody or antigen-binding fragment thereof, a Fab, asingle-chain variable fragment (scFv), or a single-domain antibody. Incertain embodiments, the antigen binding domain is a single-chainvariable fragment (scFv) comprising an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

Tolerable variations of the antigen binding domain sequences will beknown to those of skill in the art. For example, in some embodiments theantigen-binding domain capable of binding prostate stem cell antigen(PSCA) comprises an amino acid sequence that has at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity toany of the amino acid sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5,6, 7, 8, or 9.

In some embodiments, a CAR of the present disclosure may have affinityfor one or more target antigens on one or more target cells. In someembodiments, a CAR may have affinity for one or more target antigens ona target cell. In such embodiments, the CAR is a bispecific CAR, or amultispecific CAR. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for one or moretarget antigens. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for the same targetantigen. For example, a CAR comprising one or more target-specificbinding domains having affinity for the same target antigen could binddistinct epitopes of the target antigen. When a plurality oftarget-specific binding domains is present in a CAR, the binding domainsmay be arranged in tandem and may be separated by linker peptides. Forexample, in a CAR comprising two target-specific binding domains, thebinding domains are connected to each other covalently on a singlepolypeptide chain, through an oligo- or polypeptide linker, an Fc hingeregion, or a membrane hinge region.

In certain aspects, the invention includes a bispecific CAR comprisingan extracellular domain comprising an antigen-binding domain capable ofbinding prostate stem cell antigen (PSCA) and an antigen-binding domaincapable of binding prostate specific membrane antigen (PSMA), atransmembrane domain, and an intracellular domain.

In certain embodiments, the antigen binding domain capable of bindingPSCA comprises a first heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a first lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6). The antigen binding domain capable ofbinding PSMA comprises a second heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence SNWIG (SEQ ID NO: 28),HCDR2 comprises the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO:29), and HCDR3 comprises the amino acid sequence QTGFLWSFDL (SEQ ID NO:30); and a second light chain variable region that comprises three lightchain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence RASQDISSALA (SEQ ID NO: 31), LCDR2comprises the amino acid sequence DASSLES (SEQ ID NO: 32), and LCDR3comprises the amino acid sequence QQFNSYPLT (SEQ ID NO: 33).

In certain embodiments, the antigen binding domain capable of bindingPSCA comprises a first heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a first lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIEW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6) and the antigen binding domain capableof binding PSMA comprises a second heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence EYTIH (SEQ ID NO: 68),HCDR2 comprises the amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO:69), HCDR3 comprises the amino acid sequence GWNFDY (SEQ ID NO: 70); anda second light chain variable region that comprises three light chaincomplementarity determining regions (LCDRs), wherein LCDR1 comprises theamino acid sequence KASQDVGTAVD (SEQ ID NO: 71), LCDR2 comprises theamino acid sequence WASTRHT (SEQ ID NO: 72), and LCDR3 comprises theamino acid sequence QQYNSYPLT (SEQ ID NO: 73).

In certain embodiments, the bispecific CAR comprises a first heavy chainvariable region comprising an amino acid sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 7 and a first light chain variable region comprising an aminoacid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 8 and/or a second heavy chainvariable region comprising an amino acid sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 34 and a second light chain variable region comprising anamino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35.

In certain embodiments, the bispecific CAR comprises a first heavy chainvariable region comprising an amino acid sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 7 and a first light chain variable region comprising an aminoacid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 8 and/or a second heavy chainvariable region comprising an amino acid sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 75 and a second light chain variable region comprising anamino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77.

In certain embodiments, the antigen binding domain capable of bindingPSCA is a single-chain variable fragment (scFv) comprising an amino acidsequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 9 and/or the antigen-binding domaincapable of binding PSMA is a single-chain variable fragment (scFv)comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36, 78,84, or 85.

In certain embodiments, the bispecific CAR comprises an extracellulardomain comprising an antigen-binding domain capable of binding PSCA andan antigen-binding domain capable of binding PSMA, wherein theextracellular domain comprises the amino acid sequence at least at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to identical to the amino acid sequence set forth in SEQ IDNO: 50 or 52.

In certain embodiments, the antigen binding domain capable of bindingPSCA and the antigen-binding domain capable of binding PSMA areseparated by a linker. Any linker known in the art may be used toseparate the antigen-binding domain capable of binding PSCA and theantigen-binding domain capable of binding PSMA. In certain embodiments,the linker comprises the amino acid sequence set forth in SEQ ID NO: 37or 38.

Additional PSMA binders and CARS are described in PCT/US2019/020729,contents of which are incorporated by reference in their entiretyherein. In certain embodiments, the antigen binding domain capable ofbinding PSMA comprises a heavy chain variable region comprising an aminoacid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 75. In certain embodiments,the antigen binding domain capable of binding PSMA comprises a lightchain variable region comprising an amino acid sequence at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 77. In certain embodiments, the antigen-binding domaincapable of binding PSMA is a single-chain variable fragment (scFv)comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36, 78, or84.

In certain embodiments, the antigen binding domain capable of bindingPSMA comprises any of the heavy and light chain variable regionsdisclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1, thedisclosures of which are hereby incorporated herein by reference intheir entirety. For example, an antigen binding domain capable ofbinding PSMA can comprise an scFv comprising any of the heavy and lightchain variable regions disclosed therein. Accordingly, a bispecific CARor PSMA-CAR of the present invention comprises an antigen binding domaincapable of binding PSMA can comprise any scFv and any heavy and lightchain variable regions as disclosed in WO2017212250A1 andWO2018033749A1.

In certain embodiments, an antigen binding domain capable of bindingPSMA can comprise a heavy chain variable region and a light chainvariable region of any of those set forth in Table 1:

TABLE 1 Heavy Chain Variable Region SequencesLight Chain Variable Region Sequences VH Consensus SequenceVL Consensus Sequence SEQ ID NO: 101 SEQ ID NO: 102EVQLVQSGX₁EX₂KKPGASVKVSCKX₃ DIX₁MTQSPSX₂LSASVGDRVTITCKASQDVSGYTFTEYTIHWVX₄QAX₅GKGLEWIG GTAVDWYQQKPGQAPKLLIYWASTRHTGNINPNX₆GGTTYNQKFEDRX₇TX₈TVD VPDRFX₃GSGSGTDFTLTISRLQX₄EDFAX₅YKSTSTAYMELSSLRSEDTAVYYCAAG X₆CQQYNSYPLTFGQGTX₇VDIK WNFDYWGQGTTVTVSSwherein: wherein: X₁ is Q or V; X₁ is A or P; X₂ is T or F;X₂ is V or L; X₃ is S or T; X₃ is A or T; X₄ is P or S; X₄ is R or K;X₅ is V or D; X₅ is P or H; X₆ is Y or F; and X₆ is N or Q;X₇ is K or M. X₇ is V or A; and X₈ is I or L. SEQ ID NO: 103SEQ ID NO: 104 EVQLVQSGPELKKPGASVKVSCKTSG DIVMTQSPSFLSASVGDRVTITCKASQDVGYTFTEYTIHWVKQAHGKGLEWIGNIN TAVDWYQQKPGQAPKLLIYWASTRHTGVPNNGGTTYNQKFEDRATLTVDKSTST PDRFTGSGSGTDFTLTISRLQSEDFADYFCQAYMELSSLRSEDTAVYYCAAGWNFD QYNSYPLTFGQGTMVDIK YWGQGTTVTVSS SEQ ID NO: 105SEQ ID NO: 106 EVQLVQSGAEVKKPGASVKVSCKTSG DIVMTQSPSTLSASVGDRVTITCKASQDVGYTFTEYTIHWVKQAPGKGLEWIGNIN TAVDWYQQKPGQAPKLLIYWASTRHTGVPNNGGTTYNQKFEDRATITVDKSTST PDRFTGSGSGTDFTLTISRLQSEDFADYFCQAYMELSSLRSEDTAVYYCAAGWNFD QYNSYPLTFGQGTKVDIK YWGQGTTVTVSS SEQ ID NO: 107SEQ ID NO: 108 EVQLVQSGAEVKKPGASVKVSCKTSG DIVMTQSPSTLSASVGDRVTITCKASQDVGYTFTEYTIHWVRQAPGKGLEWIGNIN TAVDWYQQKPGQAPKLLIYWASTRHTGVPNNGGTTYNQKFEDRATITVDKSTST PDRFSGSGSGTDFTLTISRLQPEDFADYYCQAYMELSSLRSEDTAVYYCAAGWNFD QYNSYPLTFGQGTKVDIK YWGQGTTVTVSS SEQ ID NO: 75SEQ ID NO: 77 EVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSTLSASVGDRVTITCKASQDVGGYTFTEYTIHWVRQAPGKGLEWIGNI TAVDWYQQKPGQAPKLLIYWASTRHTGVNPNNGGTTYNQKFEDRVTITVDKSTS PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQTAYMELSSLRSEDTAVYYCAAGWNF QYNSYPLTFGQGTKVDIK DYWGQGTTVTVSSSEQ ID NO: 109 EVQLVQSGAEVKKPGASVKVSCKAS GYTFTEYTIHWVRQAPGKGLEWIGNINPNQGGTTYNQKFEDRVTITVDKSTS TAYMELSSLRSEDTAVYYCAAGWNF DYWGQGTTVTVSSVH Consensus Sequence VL Consensus Sequence SEQ ID NO: 110SEQ ID NO: 111 EVQLVQSGX₁EX₂KKPGASVKVSCKX₃DIX₁MTQSPSX₂LSASVGDRVTITCKASQDV SGYTFTEYTIHWVX₄QAX₅GKGLEWIGGTAVDWYQQKPGQAPKLLIYWASTRHTG NINPNX₆GGTTYNQKFEDRX₇TX₈TVDVPDRFX₃GSGSGTDFTLTISRLQX₄EDFAX₅Y KSTSTAYMELSSX₉RSEDTAVYYCAX₁₀X₆CQQX₇X₈X₉X₁₀X₁₁LTFGQGTX₁₂VDIK X₁₁X₁₂X₁₃X₁₄DYWGQGTTVTVSS wherein:wherein: X₁ is Q or V; X₁ is A or P; X₂ is T or F; X₂ is V or L;X₃ is S or T; X₃ is A or T; X₄ is P or S; X₄ is R or K; X₅ is V or D;X₅ is P or H; X₆ is Y or F; X₆ is N or Q; X₇-X₁₁ is FTRYP or YNAYS; andX₇ is V or A; X₁₂ is K or M. X₈ is I or L; X₉ is L or P; andX₁₀-X₁₄ is AYWLF, GGWTF, or GAWTM. SEQ ID NO: 112 SEQ ID NO: 113EVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSTLSASVGDRVTITCKASQDVGGYTFTEYTIHWVRQAPGKGLEWIGNI TAVDWYQQKPGQAPKLLIYWASTRHTGVNPNNGGTTYNQKFEDRVTITVDKSTS PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQTAYMELSSLRSEDTAVYYCAAYWLF QYNSYPLTFGQGTKVDIK DYWGQGTTVTVSSSEQ ID NO: 114 SEQ ID NO: 115 EVQLVQSGAEVKKPGASVKVSCKASDIQMTQSPSTLSASVGDRVTITCKASQDVG GYTFTEYTIHWVRQAPGKGLEWIGNITAVDWYQQKPGQAPKLLIYWASTRHTGV NPNNGGTTYNQKFEDRVTITVDKSTSPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQ TAYMELSSLRSEDTAVYYCAGGWTFQFTRYPLTFGQGTKVDIK DYWGQGTTVTVSS SEQ ID NO: 116 SEQ ID NO: 117EVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSTLSASVGDRVTITCKASQDVGGYTFTEYTIHWVRQAPGKGLEWIGNI TAVDWYQQKPGQAPKLLIYWASTRHTGVNPNNGGTTYNQKFEDRVTITVDKSTS PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQTAYMELSSLRSEDTAVYYCAGAWTM QYNAYSLTFGQGTKVDIK DYWGQGTTVTVSSSEQ ID NO: 118 EVQLVQSGAEVKKPGASVKVSCKAS GYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTS TAYMELSSPRSEDTAVYYCAAGWNF DYWGQGTTVTVSS

Transmembrane Domain

CARs of the present invention (including bispecific CARs) may comprise atransmembrane domain that connects the antigen binding domain of the CARto the intracellular domain of the CAR. The transmembrane domain of asubject CAR is a region that is capable of spanning the plasma membraneof a cell (e.g., an immune cell or precursor thereof). The transmembranedomain is for insertion into a cell membrane, e.g., a eukaryotic cellmembrane. In some embodiments, the transmembrane domain is interposedbetween the antigen binding domain and the intracellular domain of aCAR.

In some embodiments, the transmembrane domain is naturally associatedwith one or more of the domains in the CAR. In some embodiments, thetransmembrane domain can be selected or modified by one or more aminoacid substitutions to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein, e.g., a Type Itransmembrane protein. Where the source is synthetic, the transmembranedomain may be any artificial sequence that facilitates insertion of theCAR into a cell membrane, e.g., an artificial hydrophobic sequence.Examples of the transmembrane domain of particular use in this inventioninclude, without limitation, transmembrane domains derived from (i.e.comprise at least the transmembrane region(s) of) the alpha, beta orzeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40),CD137 (4-1BB), CD154 (CD40L), ICOS (CD278), Toll-like receptor 1 (TLR1),TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or a transmembranedomain derived from a killer immunoglobulin-like receptor (KIR).

In certain embodiments, the transmembrane domain comprises atransmembrane domain of CD8. In certain embodiments, the transmembranedomain of CD8 is a transmembrane domain of CD8 alpha. In certainembodiments, the transmembrane domain the transmembrane domain of CD8comprises the amino acid sequence set forth in SEQ ID NO: 11. In certainembodiments, the transmembrane domain comprises a transmembrane domainof CD28. In certain embodiments, the transmembrane domain of CD28comprises the amino acid sequence set forth in SEQ ID NO: 12. In certainembodiments, the transmembrane domain comprises a transmembrane domainof ICOS. In certain embodiments, the transmembrane domain of ICOScomprises the amino acid sequence set forth in SEQ ID NO: 13.

In some embodiments, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

The transmembrane domains described herein can be combined with any ofthe antigen binding domains described herein, any of the intracellulardomains described herein, or any of the other domains described hereinthat may be included in a subject CAR.

In some embodiments, the transmembrane domain further comprises a hingeregion. A subject CAR of the present invention may also include a hingeregion. The hinge region of the CAR is a hydrophilic region which islocated between the antigen binding domain and the transmembrane domain.In some embodiments, this domain facilitates proper protein folding forthe CAR. The hinge region is an optional component for the CAR. Thehinge region may include a domain selected from Fc fragments ofantibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3regions of antibodies, artificial hinge sequences or combinationsthereof. Examples of hinge regions include, without limitation, a CD8ahinge, artificial hinges made of polypeptides which may be as small as,three glycines (Gly), as well as CH1 and CH3 domains of IgGs (such ashuman IgG4).

In some embodiments, a subject CAR of the present disclosure (includinga bispecific CAR) includes a hinge region that connects the antigenbinding domain with the transmembrane domain, which, in turn, connectsto the intracellular domain. The hinge region is preferably capable ofsupporting the antigen binding domain to recognize and bind to thetarget antigen on the target cells (see, e.g., Hudecek et al., CancerImmunol. Res. (2015) 3(2): 125-135). In some embodiments, the hingeregion is a flexible domain, thus allowing the antigen binding domain tohave a structure to optimally recognize the specific structure anddensity of the target antigens on a cell such as tumor cell (Hudecek etal., supra). The flexibility of the hinge region permits the hingeregion to adopt many different conformations.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. In some embodiments, the hinge region is a hinge regionpolypeptide derived from a receptor (e.g., a CD8-derived hinge region).

The hinge region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about aa, from about 20 aa to about25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa,or from about 40 aa to about 50 aa. In some embodiments, the hingeregion can have a length of greater than 5 aa, greater than 10 aa,greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa,greater than 50 aa, greater than 55 aa, or more.

Suitable hinge regions can be readily selected and can be of any of anumber of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acidsto 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitablehinge regions can have a length of greater than amino acids (e.g., 30,40, 50, 60 or more amino acids).

For example, hinge regions include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO:119) and (GGGS)_(n) (SEQ ID NO:120), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers known in the art. Glycine and glycine-serinepolymers can be used; both Gly and Ser are relatively unstructured, andtherefore can serve as a neutral tether between components. Glycinepolymers can be used; glycine accesses significantly more phi-psi spacethan even alanine, and is much less restricted than residues with longerside chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:73-142). Exemplary hinge regions can comprise amino acid sequencesincluding, but not limited to, GGSG (SEQ ID NO:122), GGSGG (SEQ IDNO:123), GSGSG (SEQ ID NO:124), GSGGG (SEQ ID NO:125), GGGSG (SEQ IDNO:126), GSSSG (SEQ ID NO:127), and the like.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. Immunoglobulin hinge region amino acid sequences are knownin the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990)87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4):1779-1789. As non-limiting examples, an immunoglobulin hinge region caninclude one of the following amino acid sequences: DKTHT (SEQ IDNO:131); CPPC (SEQ ID NO:132); CPEPKSCDTPPPCPR (SEQ ID NO:133) (see,e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503);ELKTPLGDTTHT (SEQ ID NO:134); KSCDKTHTCP (SEQ ID NO:135); KCCVDCP (SEQID NO:136); KYGPPCP (SEQ ID NO:137); EPKSCDKTHTCPPCP (SEQ ID NO:138)(human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:139) (human IgG2 hinge);ELKTPLGDTTHTCPRCP (SEQ ID NO:140) (human IgG3 hinge); SPNMVPHAHHAQ (SEQID NO:141) (human IgG4 hinge); and the like.

The hinge region can comprise an amino acid sequence of a human IgG1,IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge regioncan include one or more amino acid substitutions and/or insertionsand/or deletions compared to a wild-type (naturally-occurring) hingeregion. For example, His229 of human IgG1 hinge can be substituted withTyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP(SEQ ID NO:142); see, e.g., Yan et al., J. Biol. Chem. (2012) 287:5891-5897.

In certain embodiments, the hinge region can comprise an amino acidsequence derived from human CD8, or a variant thereof. In certainembodiments, the CAR comprises a CD8 alpha hinge sequence comprising theamino acid sequence set forth in SEQ ID NO: 10. In certain embodiments,the CAR comprises a hinge sequence comprising the amino acid sequenceset forth in SEQ ID NO: 99. In certain embodiments, the CAR comprises ahinge sequence comprising the amino acid sequence set forth in SEQ IDNO: 100.

Intracellular Domain

A subject CAR of the present invention (including a subject bispecificCAR) also includes an intracellular domain. In certain embodiments, theintracellular domain comprises a costimulatory signaling domain and anintracellular signaling domain. The intracellular domain of the CAR isresponsible for activation of at least one of the effector functions ofthe cell in which the CAR is expressed (e.g., immune cell). Theintracellular domain transduces the effector function signal and directsthe cell (e.g., immune cell) to perform its specialized function, e.g.,harming and/or destroying a target cell.

Examples of an intracellular domain for use in the invention include,but are not limited to, the cytoplasmic portion of a surface receptor,co-stimulatory molecule, and any molecule that acts in concert toinitiate signal transduction in the T cell, as well as any derivative orvariant of these elements and any synthetic sequence that has the samefunctional capability.

Examples of the intracellular domain include, without limitation, the ζchain of the T cell receptor complex or any of its homologs, e.g., ηchain, FcsRIγ and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc.,human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosinekinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn,etc.), and other molecules involved in T cell transduction, such as CD2,CD5 and CD28. In one embodiment, the intracellular signaling domain maybe human CD3 zeta chain, FcγRIII, FcsRI, cytoplasmic tails of Fcreceptors, an immunoreceptor tyrosine-based activation motif (ITAM)bearing cytoplasmic receptors, and combinations thereof.

In certain embodiments, the intracellular domain of the CAR includes anyportion of one or more co-stimulatory molecules, such as at least onesignaling domain from CD2, CD3, CD8, CD27, CD28, ICOS (CD278), 4-1BB,PD-1, any derivative or variant thereof, any synthetic sequence thereofthat has the same functional capability, and any combination thereof theintracellular domain comprises a costimulatory domain of a proteinselected from the group consisting of proteins in the TNFR superfamily,CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10,DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30,CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or anintracellular domain derived from a killer immunoglobulin-like receptor(KIR).

Further, variant intracellular signaling domains suitable for use in asubject CAR are known in the art. The YMFM motif is found in ICOS and isa SH2 binding motif that recruits both p85 and p50alpha subunits ofPI3K, resulting in enhanced AKT signaling. See, e.g., Simpson et al.(2010) Curr. Opin. Immunol., 22:326-332. In one embodiment, a CD28intracellular domain variant may be generated to comprise a YMFM motif.The YMNM motif is found in the CD28 cytoplasmic domain and is a knownbinding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. See,Harada et al. (2003) J. Exp. Med., 197(2):257-262. In one embodiment, anICOS intracellular domain variant may be generated to comprise a YMNMmotif.

In certain embodiments, the intracellular domain comprises acostimulatory domain of 4-1BB. In certain embodiments, the costimulatorydomain of 4-1BB comprises the amino acid sequence set forth in SEQ IDNO: 14. In certain embodiments, the intracellular domain comprises acostimulatory domain of CD28. In certain embodiments, the costimulatorydomain of CD28 comprises the amino acid sequence set forth in SEQ ID NO:15. In certain embodiments, the intracellular domain comprises acostimulatory domain of ICOS. In certain embodiments, the costimulatorydomain of ICOS comprises the amino acid sequence set forth in SEQ ID NO:16. In certain embodiments, the intracellular domain comprises acostimulatory domain of ICOS(YMNM). In certain embodiments, thecostimulatory domain of ICOS(YMNM) comprises the amino acid sequence setforth in SEQ ID NO: 17.

Other examples of the intracellular domain include a fragment or domainfrom one or more molecules or receptors including, but not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon RIb), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40,CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory moleculesdescribed herein, any derivative, variant, or fragment thereof, anysynthetic sequence of a co-stimulatory molecule that has the samefunctional capability, and any combination thereof.

Additional examples of intracellular domains include, withoutlimitation, intracellular signaling domains of several types of variousother immune signaling receptors, including, but not limited to, first,second, and third generation T cell signaling proteins including CD3, B7family costimulatory, and Tumor Necrosis Factor Receptor (TNFR)superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol.(2015) 33(6): 651-653). Additionally, intracellular signaling domainsmay include signaling domains used by NK and NKT cells (see, e.g.,Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signalingdomains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012)189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol.(2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

In certain embodiments, the intracellular domain comprises anintracellular signaling domain selected from the group consisting ofcytoplasmic signaling domains of a human CD3 zeta chain (CD3), FcγRIII,FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptortyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCRzeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, and CD66d, or a variant thereof. In certain embodiments, theintracellular domain comprises an intracellular domain of CD3ζ or avariant thereof. In certain embodiments, the intracellular domain ofCD3ζ comprises the amino acid sequence set forth in 18 or 19.

Intracellular domains suitable for use in a subject CAR of the presentinvention include any desired signaling domain that provides a distinctand detectable signal (e.g., increased production of one or morecytokines by the cell; change in transcription of a target gene; changein activity of a protein; change in cell behavior, e.g., cell death;cellular proliferation; cellular differentiation; cell survival;modulation of cellular signaling responses; etc.) in response toactivation of the CAR (i.e., activated by antigen and dimerizing agent).In some embodiments, the intracellular domain includes at least one(e.g., one, two, three, four, five, six, etc.) ITAM motif as describedbelow. In some embodiments, the intracellular domain includes DAP10/CD28type signaling chains. In some embodiments, the intracellular domain isnot covalently attached to the membrane bound CAR, but is insteaddiffused in the cytoplasm.

Intracellular domains suitable for use in a subject CAR of the presentinvention include immunoreceptor tyrosine-based activation motif(ITAM)-containing intracellular signaling polypeptides. In someembodiments, an ITAM motif is repeated twice in an intracellular domain,where the first and second instances of the ITAM motif are separatedfrom one another by 6 to 8 amino acids. In one embodiment, theintracellular domain of a subject CAR comprises 3 ITAM motifs.

In some embodiments, intracellular domains includes the signalingdomains of human immunoglobulin receptors that contain immunoreceptortyrosine based activation motifs (ITAMs) such as, but not limited to,FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g.,Gillis et al., Front. Immunol. (2014) 5:254).

A suitable intracellular domain can be an ITAM motif-containing portionthat is derived from a polypeptide that contains an ITAM motif. Forexample, a suitable intracellular domain can be an ITAM motif-containingdomain from any ITAM motif-containing protein. Thus, a suitableintracellular domain need not contain the entire sequence of the entireprotein from which it is derived. Examples of suitable ITAMmotif-containing polypeptides include, but are not limited to: DAP12,FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptorcomplex-associated protein alpha chain).

In one embodiment, the intracellular domain is derived from DAP12 (alsoknown as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP;PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO proteintyrosine kinase-binding protein; killer activating receptor associatedprotein; killer-activating receptor-associated protein; etc.). In oneembodiment, the intracellular domain is derived from FCER1G (also knownas FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain;fc-epsilon RI-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulinepsilon receptor subunit gamma; immunoglobulin E receptor, highaffinity, gamma chain; etc.). In one embodiment, the intracellulardomain is derived from T-cell surface glycoprotein CD3 delta chain (alsoknown as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta;CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain;T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 deltachain; etc.). In one embodiment, the intracellular domain is derivedfrom T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e,T-cell surface antigen T3/Leu-4 epsilon chain, T-cell surfaceglycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.).In one embodiment, the intracellular domain is derived from T-cellsurface glycoprotein CD3 gamma chain (also known as CD3G, T-cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). In one embodiment, the intracellular domain is derivedfrom T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z,T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ,etc.). In one embodiment, the intracellular domain is derived from CD79A(also known as B-cell antigen receptor complex-associated protein alphachain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membraneglycoprotein; ig-alpha; membrane-bound immunoglobulin-associatedprotein; surface IgM-associated protein; etc.). In one embodiment, anintracellular domain suitable for use in an FN3 CAR of the presentdisclosure includes a DAP10/CD28 type signaling chain. In oneembodiment, an intracellular domain suitable for use in an FN3 CAR ofthe present disclosure includes a ZAP70 polypeptide. In someembodiments, the intracellular domain includes a cytoplasmic signalingdomain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, theintracellular domain in the CAR includes a cytoplasmic signaling domainof human CD3 zeta.

While usually the entire intracellular domain can be employed, in manycases it is not necessary to use the entire chain. To the extent that atruncated portion of the intracellular domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The intracellular domainincludes any truncated portion of the intracellular domain sufficient totransduce the effector function signal.

The intracellular domains described herein can be combined with any ofthe antigen binding domains described herein, any of the transmembranedomains described herein, or any of the other domains described hereinthat may be included in the CAR.

Tolerable variations of the individual CAR domain sequences (hinge,transmembrane, and intracellular domains) will be known to those ofskill in the art. For example, in some embodiments the CAR domaincomprises an amino acid sequence that has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to any of theamino acid sequences set forth in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 99, and 100.

In one aspect, the invention provides a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA), comprising anantigen-binding domain, a transmembrane domain, and an intracellulardomain, wherein the antigen-binding domain comprises a heavy chainvariable region comprising an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7; and alight chain variable region comprising an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

In one aspect, the invention provides a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA), comprising anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 9.

In one aspect, the invention provides a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA), comprising anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 21, 23, 25, or 27. Tolerable variations ofthe CAR sequences will be known to those of skill in the art. Forexample, in some embodiments the CAR comprises an amino acid sequencethat has at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to any of the amino acid sequences set forthin SEQ ID NO: 21, 23, 25, or 27.

In one aspect, the invention provides a chimeric antigen receptor (CAR)capable of binding prostate specific membrane antigen (PSMA), comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 67, 87, 89, 91, 93, 95, or 97. Tolerablevariations of the CAR sequences will be known to those of skill in theart. For example, in some embodiments the CAR comprises an amino acidsequence that has at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence setforth in SEQ ID NO: 67, 87, 89, 91, 93, 95, or 97.

In one aspect, the invention provides a bispecific chimeric antigenreceptor (CAR) comprising an extracellular domain comprising anantigen-binding domain capable of binding prostate stem cell antigen(PSCA) and an antigen-binding domain capable of binding prostatespecific membrane antigen (PSMA), a transmembrane domain, and anintracellular domain.

In one aspect, the invention provides a bispecific CAR, wherein theextracellular domain comprises an antigen-binding domain capable ofbinding PSCA and an antigen-binding domain capable of binding PSMA,wherein the bispecific CAR comprises the amino acid sequence set forthin SEQ ID NO: 40, 42, or 80-82. Tolerable variations of the CARsequences will be known to those of skill in the art. For example, insome embodiments the CAR comprises an amino acid sequence that has atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to any of the amino acid sequences set forth in SEQ IDNO: 40, 42, or 80-82.

In one aspect, the invention provides a bispecific chimeric antigenreceptor (CAR) capable of binding prostate stem cell antigen (PSCA) andprostate specific membrane antigen (PSMA), wherein the antigen bindingdomain capable of binding PSMA is a single-chain variable fragment(scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78, 84, or 85 and/orthe antigen-binding domain capable of binding PSCA and anantigen-binding domain capable of binding PSMA comprises the amino acidsequence set forth in SEQ ID NO: 50 or 52.

In one aspect, the invention provides a bispecific chimeric antigenreceptor (CAR) capable of binding prostate stem cell antigen (PSCA) andprostate specific membrane antigen (PSMA), comprising extracellulardomain comprising an antigen binding domain capable of PSCA and anantigen-binding domain capable of binding PSMA, a transmembrane domain,and an intracellular domain. The antigen binding domain capable ofbinding PSCA comprises a heavy chain variable region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 7, and/or a light chain variable regioncomprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 8. The antigen-binding domaincapable of binding PSMA comprises a heavy chain variable regioncomprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 34, and/or a light chainvariable region comprising an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35.

In one aspect, the invention provides a bispecific chimeric antigenreceptor (CAR) capable of binding prostate stem cell antigen (PSCA) andprostate specific membrane antigen (PSMA), wherein the antigen bindingdomain capable of binding PSCA is a single-chain variable fragment(scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and/or the antigenbinding domain capable of binding PSMA is a single-chain variablefragment (scFv) comprising an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36.

In one aspect, the invention provides a bispecific chimeric antigenreceptor (CAR) capable of binding prostate stem cell antigen (PSCA) andprostate specific membrane antigen (PSMA), comprising an extracellulardomain comprising an antigen binding domain capable of PSCA and anantigen-binding domain capable of binding PSMA, a transmembrane domain,and an intracellular domain. The antigen binding domain capable ofbinding PSCA comprises a heavy chain variable region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 7, and a light chain variable region comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 8. The antigen-binding domain capable ofbinding PSMA comprises a heavy chain variable region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 75, and a light chain variable region comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 77.

In one aspect, the invention provides a bispecific CAR, wherein theextracellular domain comprises an antigen binding domain capable ofbinding PSCA comprising a first heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence DYYIH (SEQ ID NO: 1),HCDR2 comprises the amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence TGGF (SEQ ID NO: 3);and/or a first light chain variable region that comprises three lightchain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2comprises the amino acid sequence DTSKLAS (SEQ ID NO: 5), and LCDR3comprises the amino acid sequence QQWSSSPFT (SEQ ID NO: 6); and anantigen binding domain capable of binding PSMA comprising a second heavychain variable region that comprises three heavy chain complementaritydetermining regions (HCDRs), wherein HCDR1 comprises the amino acidsequence SNWIG (SEQ ID NO: 28), HCDR2 comprises the amino acid sequenceIIYPGDSDTRYSPSFQG (SEQ ID NO: 29), and HCDR3 comprises the amino acidsequence QTGFLWSFDL (SEQ ID NO: 30); and/or a second light chainvariable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence RASQDISSALA (SEQ ID NO: 31), LCDR2 comprises the amino acidsequence DASSLES (SEQ ID NO: 32), and LCDR3 comprises the amino acidsequence QQFNSYPLT (SEQ ID NO: 33).

In one aspect, the invention provides a bispecific CAR, wherein theextracellular domain comprises an antigen binding domain capable ofbinding PSCA comprising a first heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence DYYIH (SEQ ID NO: 1),HCDR2 comprises the amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence TGGF (SEQ ID NO: 3);and/or a first light chain variable region that comprises three lightchain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2comprises the amino acid sequence DTSKLAS (SEQ ID NO: 5), and LCDR3comprises the amino acid sequence QQWSSSPFT (SEQ ID NO: 6); and theantigen binding domain capable of binding PSMA comprises a second heavychain variable region that comprises three heavy chain complementaritydetermining regions (HCDRs), wherein HCDR1 comprises the amino acidsequence EYTIH (SEQ ID NO: 68), HCDR2 comprises the amino acid sequenceNINPNNGGTTYNQKFED (SEQ ID NO: 69), and HCDR3 comprises the amino acidsequence GWNFDY (SEQ ID NO: 70); and/or a second light chain variableregion that comprises three light chain complementarity determiningregions (LCDRs), wherein LCDR1 comprises the amino acid sequenceKASQDVGTAVD (SEQ ID NO: 71), LCDR2 comprises the amino acid sequenceWASTRHT (SEQ ID NO: 72), and LCDR3 comprises the amino acid sequenceQQYNSYPLT (SEQ ID NO: 73).

TABLE 2 Sequences used in the invention SEQ ID NO: NameAmino Acid/Nucleotide Sequence 1 PSCA 2B3 HCDR1 DYYIH 2 PSCA 2B3 HCDR2WIDPENGDTEFVPKFQG 3 PSCA 2B3 HCDR3 TGGF 4 PSCA 2B3 LCDR1 SASSSVRFIHW 5PSCA 2B3 LCDR2 DTSKLAS 6 PSCA 2B3 LCDR3 QQWSSSPFT 7 PSCA 2B3 VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAED TAVYYCKTGGFWGQGTLVTVSS 8PSCA 2B3 VL DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSP FTFGQGTKVEIK 9PSCA 2B3 scFv DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQ GTLVTVSS 10 CD8 alpha hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 99 HingeNHDASAATTNTGAHHASQPLSLRPEACRPAAGGAVHTRGLDFAC D 100 HingeHTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 11 CD8 alphaIYIWAPLAGTCGVLLLSLVITLY transmembrane domain 12 CD28FWVLVVVGGVLACYSLLVTVAFIIFWV transmembrane domain 13 ICOSFWLPIGCAAFVVVCILGCILI transmembrane domain 14 4-1BB ICDKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE 15 CD28 ICDRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 16 ICOS ICDCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 17 ICOS(YMNM) ICDCWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTL 18 CD3 zeta ICDRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR19 CD3 zeta (Q14K) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE ICDMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR 20PSCA CARatggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatcc2B3.BBZagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagcttataacgagctcaatctaggacgaagagaggagtacgacgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 21 PSCA CAR MALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS 2B3.BBZSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 22 PSCA CARAtggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatc2B3.28ZcagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccagggggggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcAccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatTtttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacggacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagcttataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 23 PSCA CARMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS 2B3.28ZSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 24 PSCA CARatggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatcc2B3.ICOSZagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcAccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatTtctggttacccataggatgtgcagcctttgttgtagtctgcattttgggatgcatacttatttgttggcttacaaaaaagaagtattcatccagtgtgcacgaccctaacggtgaatacatgttcatgagagcagtgaacacagccaaaaaatcCagactcacagatgtgaccctaAgagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 25 PSCA CARMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS 2B3.ICOSZSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR26 PSCA CARAtggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatc2B3.ICOS.cagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcYMNM.ZgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcAccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttctggttacccataggatgtgcagcctttgttgtagtctgcattttgggatgcatacttatttgttggcttacaaaaaagaagtattcatccagtgtgcacgaccctaacggtgaatacatgAACatgagagcagtgaacacagccaaaaaatcCagactcacagatgtgaccctaagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 27 PSCA CARMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS 2B3.ICOS.SSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTL YMNM.ZTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR28 PSMA 2F5 HCDR1 SNWIG 29 PSMA 2F5 HCDR2 IIYPGDSDTRYSPSFQG 30PSMA 2F5 HCDR3 QTGFLWSFDL 31 PSMA 2F5 LCDR1 RASQDISSALA 32PSMA 2F5 LCDR2 DASSLES 33 PSMA 2F5 LCDR3 QQFNSYPLT 34 PSMA 2F5 VHEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSS 35 PSMA 2F5 VLAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIKIK 36PSMA 2F5 scFv EVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIKIK 37Linker GGGGS 38 Linker GGGGGSGGGGSGGGGSGGGGS 39 2B3-L-2F5.BBZatggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatccCARagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcggaggtggtggatccggcggagggggaagtggcggggggggtccggcggcggcggctcggaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacagttttaccagcaactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagacaaactggtttcctctggtccttcgatctctggggccgtggcaccctggtcactgtctcctcaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggacattagcagtgctttagcctggtatcagcagaaaccggggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttacccgctcactttcggcggagggaccaaggtggagatcaaaatcaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 40 2B3-L-2F5.BBZMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS CARSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPBACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR 412B3-S-2F5.BBZatggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatccCARagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcggaggtggtggatccgaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacagttttaccagcaactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagacaaactggtttcctctggtccttcgatctctggggccgtggcaccctggtcactgtctcctcaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggacattagcagtgctttagcctggtatcagcagaaaccggggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttacccgctcactttcggcggagggaccaaggtggagatcaaaatcaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 42 2B3-S-2F5.BBZMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSAS CARSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR 43 PSCA 2B3 VHgaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagt 44PSCA 2B3 VLgatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaag 45 PSCA 2B3 scFvgatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagt 46 PSMA 2F5 VHGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTG TCTCCTCA 47 PSMA 2F5 VLGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAAATCAAAA 48PSMA 2F5 scFv GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTQGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAA 49 2B3-L-2F5gatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagExtracellulartgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatdomainctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcggaggtggtggatccggcggagggggaagtggcggggggggtccggcggcggcggctcggaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacagttttaccagcaactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagacaaactggtttcctctggtccttcgatctctggggccgtggcaccctggtcactgtctcctcaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggacattagcagtgctttagcctggtatcagcagaaaccggggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttacccgctcactttcggcggagggaccaaggtggagatcaaaatcaaaa 50 2B3-L-2FSDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLI ExtracellularYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSP domainFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKI K 51 2B3-S-2F5gatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagExtracellulartgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatdomainctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcggaggtggtggatccgaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacagttttaccagcaactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtggaacagcctgaaggcctcggacaccgccatgtattactgtgcgagacaaactggtttcctctggtccttcgatctctggggccgtggcaccctggtcactgtctcctcaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggacattagcagtgctttagcctggtatcagcagaaaccggggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttacccgctcactttcggcggagggaccaaggtggagatcaaaatcaaaa 522B3-S-2F5 DIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLI ExtracellularYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSP domainFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIK 53 linker GGCGGCGGCGGCAGC 54 linkerGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGC 55TGFBRII.dnatgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgtggacgcgtatcgccagcacgatcccaccgcacgttcagaagtcggttaataacgacatgatagtcactgacaacaacggtgcagtcaagtttccacaactgtgtaaattttgtgatgtgagattttccacctgtgacaaccagaaatcctgcatgagcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtggctgtatggagaaagaatgacgagaacataacactagagacagtttgccatgaccccaagctcccctaccatgactttattctggaagatgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtgagactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatcatcttctcagaagaatataacaccagcaatcctgacttgttgctagtcatatttcaagtgacaggcatcagcctcctgccaccactgggagttgccatatctgtcatcatcatcttctactgctaccgcgttaaccggcagcagaagctgagttcatccgga 56 TGFBRII.dnMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIF YCYRVNRQQKLSSSG 57PD1-CTM-CD28atgcagatcccacaggcgccctggccagtcgtctgggcggtgctacaactgggctggcggccaggatgswitchgttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccagttccaaaccctggtgttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc 58 PD1-CTM-CD28MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVV switchTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRS 59hIFNgR-IL12rb1atggctctcctctttctcctaccccttgtcatgcagggtgtgagcagggctgagatgggcaccgcggatctgswitchgggccgtcctcagtgcctacaccaactaatgttacaattgaatcctataacatgaaccctatcgtatattgggagtaccagatcatgccacaggtccctgtttttaccgtagaggtaaagaactatggtgttaagaattcagaatggattgatgcctgcatcaatatttctcatcattattgtaatatttctgatcatgttggtgatccatcaaattctctttgggtcagagttaaagccagggttggacaaaaagaatctgcctatgcaaagtcagaagaatttgctgtatgccgagatggaaaaattggaccacctaaactggatatcagaaaggaggagaagcaaatcatgattgacatatttcacccttcagtttttgtaaatggagacgagcaggaagtcgattatgatcccgaaactacctgttacattagggtgtacaatgtgtatgtgagaatgaacggaagtgagatccagtataaaatactcacgcagaaggaagatgattgtgacgagattcagtgccagttagcgattccagtatcctcactgaattctcagtactgtgtttcagcagaaggagtcttacatgtgtggggtgttacaactgaaaagtcaaaagaagtttgtattaccattttcaatagcagtataaaaggttctctitggattccagttgttgctgctttactactctttctagtgcttagcctggtattcatcagggccgcacggcacctgtgcccgccgctgcccacaccctgtgccagctccgccattgagttccctggagggaaggagacttggcagtggatcaacccagtggacttccaggaagaggcatccctgcaggaggccctggtggtagagatgtcctgggacaaaggcgagaggactgagcctctcgagaagacagagctacctgagggtgcccctgagctggccctggatacagagttgtccttggaggatggagacaggtgcaaggccaagatgtga 60hIFNgR-IL12rb1 MALLFLLPLVMQGVSRAEMGTADLGPSSVPTPTNVTIESYNMNPIV switchYWEYQIMPQVPVFTVEVKNYGVKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKARVGQKESAYAKSEEFAVCRDGKIGPPKLDIRKEEKQIMIDIFHPSVFVNGDEQEVDYDPETTCYIRVYNVYVRMNGSEIQYKILTQKEDDCDEIQCQLAIPVSSLNSQYCVSAEGVLHVWGVTTEKSKEVCITIFNSSIKGSLWIPVVAALLLFLVLSLVFIRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM 61 hIFNgb-IL12rb2atgcgaccgacgctgctgtggtcgctgctgctgctgctcggagtcttcgccgccgccgccgcggccccgswitchccagaccctctttcccagctgcccgctcctcagcacccgaagattcgcctgtacaacgcagagcaggtcctgagttgggagccagtggccctgagcaatagcacgaggcctgttgtctaccaagtgcagtttaaatacaccgacagtaaatggttcacggccgacatcatgtccataggggtgaattgtacacagatcacagcaacagagtgtgacttcactgccgccagtccctcagcaggcttcccaatggatttcaatgtcactctacgccttcgagctgagctgggagcactccattctgcctgggtgacaatgccttggtttcaacactatcggaatgtgactgtcgggcctccagaaaacattgaggtgaccccaggagaaggctccctcatcatcaggttctcctctccctttgacatcgctgatacctccacggcctttttttgttattatgtccattactgggaaaaaggaggaatccaacaggtcaaaggccctttcagaagcaactccatttcattggataacttaaaaccctccagagtgtactgtttacaagtccaggcacaactgctttggaacaaaagtaacatctttagagtcgggcatttaagcaacatatcttgctacgaaacaatggcagatgcctccactgagcttcagcaagtcatcctgatctccgtgggaacattttcgttgctgtcggtgctggcaggagcctgtttcttcctggtcctgaaatatcagcaaaaggtgtttgttctcctagcagccctcagacctcagtggtgtagcagagaaattccagatccagcaaatagcacttgcgctaagaaatatcccattgcagaggagaagacacagctgcccttggacaggctcctgatagactggcccacgcctgaagatcctgaaccgctggtcatcagtgaagtccttcatcaagtgaccccagttttcagacatcccccctgctccaactggccacaaagggaaaaaggaatccaaggtcatcaggcctctgagaaagacatgatgcacagtgcctcaagcccaccacctccaagagctctccaagctgagagcagacaactggtggatctgtacaaggtgctggagagcaggggctccgacccaaagccagaaaacccagcctgtccctggacggtgctcccagcaggtgaccttcccacccatgatggctacttaccctccaacatagatgacctcccctcacatgaggcacctctcgctgactctctggaagaactggagcctcagcacatctccctttctgttttcccctcaagttctcttcacccactcaccttctcctgtggtgataagctgactctggatcagttaaagatgaggtgtgactccctcatgctctga 62 hIFNgb-IL12rb2MRPTLLWSLLLLLGVFAAAAAAPPDPLSQLPAPQHPKIRLYNAEQ switchVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTADIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWVTMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYVHYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVGHLSNISCYETMADASTELQQVILISVGTFSLLSVLAGACFFLVLKYQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML 63 10A5-1412 (aPDL1-atgggctggagttgcatcattctcttcctcgtggcgaccgcaacaggggtgcactccgacatccagatgacaCd28 bispecificccagtccccgagttccctgtctgcttccgtgggagatcgcgtgactatcacctgccgggcttcccagggcaAb)tctcttcctggctggcgtggtaccagcagaaaccagaaaaggctcctaagtccctgatctacgcagcttcgtccctccaatccggcgtcccctctcgcttctccggctccggatccggcaccgacttcacgctgacaatctcgagtttgcagcccgaggacttcgccacctactactgccagcagtacaactcctacccttacaccttcggccagggcacaaagctcgaaatcaagtcggggggggggggtcgcaggtccagctggtgcagtccggcgccgaagtcaagaagcccggagcaagtgtgaaagtgtcgtgcaaggcaagtgggtataccttcacctcatacgacgtacactgggtgcgccaggcgcccggtcagcgccttgagtggatgggctggctccacgccgacaccggcattaccaagttctctcagaagttccagggaagagtgaccataacacgcgacaccagtgcttccacagcttacatggaactttcgagtctgagatccgaggacacagccgtgtattactgtgcccgtgagcgcatccagctgtggttcgactactgggggcagggcaccctcgtgacggtgtcgtcggggggggggggagtcaggtgcagctggtgcagtccggagccgaggtaaagaagccaggcgcttccgtcaaggtgtcatgcaaggcctcaggctacaccttcacaagctattacatccactgggtgcgccaagctcccggtcagggcttggagtggatcgggtgcatttacccagggaacgtcaacacaaactacaacgagaagttcaaggatcgggcaaccctgaccgtggacacatccatctctaccgcctacatggagctgtcacgcctgcgctctgatgacaccgcagtgtacttctgtaccaggagtcactacggcctggactggaactttgatgtctggggccagggaaccaccgtgacggtgtccagtgtggagggcggtagtggcggctctggtgggtccggaggctcaggcggcgtgatggatgacattcagatgacccagagtccctcctccctctccgcttccgtcggagaccgcgtgaccatcacttgtcacgcctcacagaatatctacgtgtggctgaactggtaccaacagaagcccggcaaggcccccaagctgcttatctataaagcgtccaacctccacacgggagtcccttcccgcttctccggatccggcagtgggacggacttcacactcacaatctcgtcgctgcagccagaggactitgcgacgtactactgccagcagggccagacctacccatatactttcggcggcgggaccaaggtggagattaag 64 10A5-1412 (aPDL1-MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQ aCd28 bispecificGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTL Ab)TISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTK VEIK 65 TGFB-1412atgggttggtcctgcatcatcctgtttctcgtggccaccgccaccggcgtgcactccgaaattgtgttgaca(aTGFbRII-aCd28cagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgtbispecific Ab)tagaagtttcttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctccgacgttcggccaagggaccaaggtggaaatcaaaagtggagggggcggttcacagctacagctgcaggagtcgggcccaggactggtgaagccttcggagaccctatccctcacctgcactgtctctggtggctccatcagcagtagtagttactcctggggctggatccgccagcccccagggaagggcctggagtggattgggagtttctattacagtgggatcacctactacagcccgtccctcaagagtcgaattatcatatccgaagacacgtccaagaaccagttctccctgaagctgagttctgtgaccgccgcagacacggctgtgtattactgtgcgagcgggtttactatgattcggggagcccttgactactggggccagggaaccctg 66 TGFB-1412MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQS (aTGFbRII-aCd28VRSFLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI bispecific Ab)SSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYSWGWIRQPPGKGLEWIGSFYYSGITYYSPSLKSRIIISEDTSKNQFSLKLSSVTAADTAVYYCASGFT MIRGALDYWGQGTL 67PSMA 2F5-BBZ MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGS CARGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 68 hJ591 HCDR1 EYTIH 69 hJ591 HCDR2NINPNNGGTTYNQKFED 70 hJ591 HCDR3 GWNFDY 71 hJ591 LCDR1 KASQDVGTAVD 72hJ591 LCDR2 WASTRHT 73 hJ591 LCDR3 QQYNSYPLT 74 hJ591 VHGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCA 75 hJ591 VHEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSED TAVYYCAAGWNFDYWGQGTTVTVSS76 hJ591 VL GACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACC AAGGTGGATATCAAA 77 hJ591 VLDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQY NSYPLTFGQGTKVDIK 78hJ591 scFv (VL- DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPK VH)LLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDY WGQGTTVTVSS 83hJ591 scFv (VL-GacattcagatgacccagtctcccagcaccctgtccgcatcagtaggagacagggtcaccatcacttgcaVH)aggccagtcaggatgtgggtactgctgtagactggtatcaacagaaaccagggcaagctcctaaactactgatttactgggcatccacccggcacactggagtccctgatcgcttcagcggcagtggatctgggacagatttcactctcaccatcagcagactgcagcctgaagactttgcagtttattactgtcagcaatataacagctatcctctcacgttcggccaggggaccaaggtggatatcaaaggaggcggaggatctggcggcggaggaagttctggcggaggcagcgaggtccagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacacattcactgaatacaccatccactgggtgaggcaggcccctggaaagggccttgagtggattggaaacattaatcctaacaatggtggtactacctacaaccagaagttcgaggacagagtcacaatcactgtagacaagtccaccagcacagcctacatggagctcagcagcctgagatctgaggatactgcagtctattactgtgcagctggttggaactttgactactggggccaaggcaccacggtcaccgtctcctca 84 hJ591 scFv (VL-DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPK VH)LLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS 85 hJ591 scFv (VH-EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKG VL)LEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNS YPLTFGQGTKVDIK 86hJ591 scFv (VH-Gaggtccagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaa VL)ggcttctggatacacattcactgaatacaccatccactgggtgaggcaggcccctggaaagggccttgagtggattggaaacattaatcctaacaatggtggtactacctacaaccagaagttcgaggacagagtcacaatcactgtagacaagtccaccagcacagcctacatggagctcagcagcctgagatctgaggatactgcagtctattactgtgcagctggttggaactttgactactggggccaaggcaccacggtcaccgtctcctcaggaggcggaggatctggcggcggaggaagttctggcggaggcagcgacattcagatgacccagtctcccagcaccctgtccgcatcagtaggagacagggtcaccatcacttgcaaggccagtcaggatgtgggtactgctgtagactggtatcaacagaaaccagggcaagctcctaaactactgatttactgggcatccacccggcacactggagtccctgatcgcttcagcggcagtggatctgggacagatttcactctcaccatcagcagactgcagcctgaagactttgcagtttattactgtcagcaatataacagctatcctctcacgttcggccaggggaccaaggtggatatcaaa 87 hJ591VHVK.BBZ MALPVTALLLPLALLLHAARPGEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 88 hJ591VHVK.BBZATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGAGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGC 89hJ591VKVH.BBZ MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 90 hJ591VKVH.BBZATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGC 91hJ591VKVH.ICOS MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCK BBZASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 92hJ591VKVH.ICOS ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCT BBZGCTGCACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 93 hJ591VKVH.ICOSMALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCK BBZYMNMASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 94hJ591VKVH.ICOS ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCT BBZYMNMGCTGCACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 95 hJ591VKVH.ICOSZMALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR96 hJ591VKVH.ICOSZ ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 97 hJ591VKVH.ICOSZMALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCK YMNMASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR98 hJ591VKVH.ICOSZ ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCT YMNMGCTGCACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 79 2B3-hJ591.BBZatggccctgcctgtgacagccctgctgctgcctctggctctgctgctgcacgccgccagacctggaGccaccatggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggggggaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcGgaggtggtggatccGACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGgaggcggaggatctggggcggaggaagttctggcggaggcagcGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAaACcacgacgccagcgccgcgaccaccaacaccggcgcccaccatgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatAtctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcAaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagacggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgAgagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgacgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaacgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgacggcctttaccagggtctcagtacagccaccaaggacacctacgagcccttcacatgcaggccctgccccctcgc 80 2B3-hJ591.BBZMALPVTALLLPLALLLHAARPGATMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSNHDASAATTNTGAHHASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 81 2B3-hJ591.BBZMALPVTALLLPLALLLHAARPGATMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSHTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR82 2B3-hJ591.BBZ MALPVTALLLPLALLLHAARPGATMALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATISADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSAAGGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR

C. Nucleic Acids and Expression Vectors

The present disclosure provides a nucleic acid encoding a CAR. Thenucleic acid of the present disclosure may comprises a polynucleotidesequence encoding any one of the CARs (including the bispecific CARs)disclosed herein.

In certain aspects, the invention includes a nucleic acid comprising apolynucleotide sequence encoding a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA), comprising anantigen binding domain, a transmembrane domain, and an intracellulardomain. In certain embodiments, the antigen-binding domain comprises: aheavy chain variable region that comprises three heavy chaincomplementarity determining regions (HCDRs), wherein HCDR1 comprises theamino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprises the amino acidsequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3 comprises the aminoacid sequence TGGF (SEQ ID NO: 3); and a light chain variable regionthat comprises three light chain complementarity determining regions(LCDRs), wherein LCDR1 comprises the amino acid sequence SASSSVRFIHW(SEQ ID NO: 4), LCDR2 comprises the amino acid sequence DTSKLAS (SEQ IDNO: 5), and LCDR3 comprises the amino acid sequence QQWSSSPFT (SEQ IDNO: 6).

In certain embodiments, the antigen binding domain comprises a heavychain variable region encoded by a polynucleotide sequence at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 43 and/or a light chain variable region encoded by apolynucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44.

In certain embodiments, the antigen binding domain is a single-chainvariable fragment (scFv) encoded by a polynucleotide sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 45.

Also provided is a nucleic acid comprising a polynucleotide sequenceencoding a chimeric antigen receptor (CAR) capable of binding prostatestem cell antigen (PSCA), comprising an antigen-binding domain, atransmembrane domain, and an intracellular domain, wherein theantigen-binding domain comprises a heavy chain variable regioncomprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7; and/ora light chain variable region comprising an amino acid sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 8.

Also provided is a nucleic acid comprising a polynucleotide sequenceencoding a chimeric antigen receptor (CAR) capable of binding prostatestem cell antigen (PSCA), comprising a polynucleotide sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 20.

The invention also provides a nucleic acid comprising a polynucleotidesequence encoding a chimeric antigen receptor (CAR) capable of bindingprostate stem cell antigen (PSCA), comprising a polynucleotide sequenceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 20, 22, 24, or 26.

The invention also provides a nucleic acid comprising a polynucleotidesequence encoding a chimeric antigen receptor (CAR) capable of bindingprostate specific membrane antigen (PSMA), wherein the PSMA-CARcomprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acidsequence set forth in SEQ ID NO: 67, 87, 89, 91, 93, 95, or 97. Incertain embodiments, the PSMA-CAR is encoded by a polynucleotidesequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 88, 90, 92, 94, 96, or 98.

Also provided is a nucleic acid comprising a polynucleotide sequenceencoding a bispecific chimeric antigen receptor (CAR) capable of bindingprostate stem cell antigen (PSCA) and prostate specific membrane antigen(PSMA), wherein the bispecific CAR comprises an extracellular domaincomprising an antigen-binding domain capable of PSCA and anantigen-binding domain capable of binding PSMA, a transmembrane domain,and an intracellular domain.

In certain embodiments, the extracellular domain comprises an antigenbinding domain capable of binding PSCA and an antigen binding domaincapable of binding PSMA. The antigen binding domain capable of bindingPSCA comprises a first heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a first lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6). The antigen binding domain capable ofbinding PSMA comprises a second heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence SNWIG (SEQ ID NO: 28),HCDR2 comprises the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO:29), and HCDR3 comprises the amino acid sequence QTGFLWSFDL (SEQ ID NO:30); and a second light chain variable region that comprises three lightchain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence RASQDISSALA (SEQ ID NO: 31), LCDR2comprises the amino acid sequence DASSLES (SEQ ID NO: 32), and LCDR3comprises the amino acid sequence QQFNSYPLT (SEQ ID NO: 33).

In certain embodiments, the extracellular domain comprises an antigenbinding domain capable of binding PSCA and an antigen binding domaincapable of binding PSMA wherein the antigen binding domain capable ofbinding PSCA comprises a first heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence DYYIH (SEQ ID NO: 1),HCDR2 comprises the amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence TGGF (SEQ ID NO: 3); anda first light chain variable region that comprises three light chaincomplementarity determining regions (LCDRs), wherein LCDR1 comprises theamino acid sequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises theamino acid sequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises theamino acid sequence QQWSSSPFT (SEQ ID NO: 6). The antigen binding domaincapable of binding PSMA comprises a second heavy chain variable regionthat comprises three heavy chain complementarity determining regions(HCDRs), wherein HCDR1 comprises the amino acid sequence EYTIH (SEQ IDNO: 68), HCDR2 comprises the amino acid sequence NINPNNGGTTYNQKFED (SEQID NO: 69), and HCDR3 comprises the amino acid sequence GWNFDY (SEQ IDNO: 70); and a second light chain variable region that comprises threelight chain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence KASQDVGTAVD (SEQ ID NO: 71), LCDR2comprises the amino acid sequence WASTRHT (SEQ ID NO: 72), and LCDR3comprises the amino acid sequence QQYNSYPLT (SEQ ID NO: 73).

In certain embodiments, the first heavy chain variable region (capableof binding PSCA) is encoded by a polynucleotide sequence at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 43; and the first light chain variable region (capable ofbinding PSCA) is encoded by a polynucleotide sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 44 and/or the second heavy chain variable region (capable ofbinding PSMA) is encoded by a polynucleotide sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 46 or 74; and the second light chain variable region (capableof binding PSMA) is encoded by a polynucleotide sequence at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 47 or 76.

In certain embodiments, the antigen binding domain capable of bindingPSCA comprises an scFv encoded by a polynucleotide sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 45. In certain embodiments, the antigen bindingdomain capable of binding PSMA comprises an scFv encoded by apolynucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48, 83, or 86.

In certain embodiments, the extracellular domain is encoded by apolynucleotide sequence comprising a nucleic acid sequence set forth inSEQ ID NO: 49 or 51.

In certain embodiments, the CAR (including the bispecific CAR) furthercomprises a hinge sequence comprising the amino acid sequence set forthin SEQ ID NO: 10, 99, or 100. In certain embodiments, the transmembranedomain of the CAR or bispecific CAR comprises a transmembrane domain ofCD8 alpha comprising the amino acid sequence set forth in SEQ ID NO: 11.In certain embodiments, the transmembrane domain comprises atransmembrane domain of CD28 comprising the amino acid sequence setforth in SEQ ID NO: 12. In certain embodiments, the transmembrane domaincomprises a transmembrane domain of ICOS comprising the amino acidsequence set forth in SEQ ID NO: 13.

In certain embodiments, the intracellular domain comprises acostimulatory signaling domain and an intracellular signaling domain. Incertain embodiments, the costimulatory signaling domain comprises acostimulatory domain of 4-1BB comprising the amino acid sequence setforth in SEQ ID NO: 14. In certain embodiments, the costimulatorysignaling domain comprises a costimulatory domain of CD28 comprising theamino acid sequence set forth in SEQ ID NO: 15. In certain embodiments,the costimulatory signaling domain comprises a costimulatory domain ofICOS comprising the amino acid sequence set forth in SEQ ID NO: 16. Incertain embodiments, the costimulatory signaling domain comprises acostimulatory domain of ICOS(YMNM) comprising the amino acid sequenceset forth in SEQ ID NO: 17. In certain embodiments, the intracellularsignaling domain comprises an intracellular domain of CD3ζ or a variantthereof, wherein the intracellular domain of CD3ζ comprises the aminoacid sequence set forth in SEQ ID NO: 18 or 19.

In certain embodiments, the bispecific CAR is encoded by apolynucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39, 41, or 79.

Another aspect of the invention includes a nucleic acid comprising afirst polynucleotide sequence encoding a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA-CAR), and a secondpolynucleotide sequence encoding a dominant negative receptor, whereinthe CAR comprises an antigen binding domain, a transmembrane domain, andan intracellular domain.

In certain embodiments, the dominant negative receptor is a truncatedvariant of a wild-type protein associated with a negative signal. Incertain embodiments, the truncated variant of a wild-type proteinassociated with a negative signal comprises an amino acid sequencesequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 56 and/or is encoded by a polynucleotidesequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 55.

Also provided is a nucleic acid comprising a first polynucleotidesequence encoding a chimeric antigen receptor (CAR) capable of bindingprostate stem cell antigen (PSCA-CAR), and a second polynucleotidesequence encoding a switch receptor, wherein the CAR comprises anantigen-binding domain, a transmembrane domain, and an intracellulardomain.

In certain embodiments, the switch receptor comprises a first domainderived from a first switch polypeptide that is associated with anegative signal; and a second domain derived from a second switchpolypeptide that is associated with a positive signal. In certainembodiments, the first domain comprises at least a portion of theextracellular domain of the first switch polypeptide that is associatedwith a negative signal, and the second domain comprises at least aportion of the intracellular domain of the second switch polypeptidethat is associated with a positive signal.

In certain embodiments, the switch receptor further comprises a switchreceptor transmembrane domain. In certain embodiments, the switchreceptor transmembrane domain comprises: the transmembrane domain of thefirst switch polypeptide that is associated with a negative signal; orthe transmembrane domain of the second switch polypeptide that isassociated with a positive signal.

In certain embodiments, the first switch polypeptide that is associatedwith a negative signal is selected from the group consisting of CTLA4,PD-1, PD-L1, BTLA, TIM-3, an IFNγR, and a TGFβR. In certain embodiments,wherein the switch polypeptide that is associated with a positive signalis selected from the group consisting of CD28, ICOS, and an IL-12R.

In certain embodiments, the switch receptor comprises a first domaincomprising at least a portion of the extracellular domain of PD1; aswitch receptor transmembrane domain comprising at least a portion ofthe transmembrane domain of CD28; and a second domain comprising atleast a portion of the intracellular domain of CD28. PD1-CD28 switchreceptors are described in Liu X, et al. (2016) Cancer research, 76(6),1578-1590, contents of which are incorporated by reference in theirentirety herein. In certain embodiments, the switch receptor is encodedby a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 57 and/or comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 58.

In certain embodiments, the switch receptor comprises a first domaincomprising at least a portion of the extracellular domain of an IFNγR;and a second domain comprising at least a portion of the intracellulardomain of IL12Rβ1. In certain embodiments, the switch receptor isencoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth inSEQ ID NO: 59 and/or comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60.

In certain embodiments, the switch receptor comprises a first domaincomprising at least a portion of the extracellular domain of an IFNγR;and a second domain comprising at least a portion of the intracellulardomain of IL12Rβ2. In certain embodiments, the switch receptor isencoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:61 and/or comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62.

Additional switch receptors are described in PCT/US2019/020729, contentsof which are incorporated by reference in their entirety herein.

Another aspect of the invention provides a nucleic acid comprising afirst polynucleotide sequence encoding a chimeric antigen receptor (CAR)capable of binding prostate stem cell antigen (PSCA-CAR), and a secondpolynucleotide sequence encoding a bispecific antibody, wherein the CARcomprises an antigen-binding domain, a transmembrane domain, and anintracellular domain.

In certain embodiments, the bispecific antibody comprises a firstbinding domain and a second binding domain. In certain embodiments, thefirst binding domain binds to a negative signal selected from the groupconsisting of CTLA4, PD-1, PD-L1, BTLA, TIM-3, and a TGFβR. In certainembodiments, the second binding domain binds to a costimulatorymolecule. In certain embodiments, the costimulatory molecule is CD28.

In certain embodiments, the bispecific antibody comprises a firstbinding domain capable of binding PD-L1, and a second binding domaincapable of binding CD28. In certain embodiments, the bispecific antibodyis encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 63 and/or comprisesan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 64.

In certain embodiments, the bispecific antibody comprises a firstbinding domain capable of binding TGFβR2, and a second binding domaincapable of binding CD28. In certain embodiments, the bispecific antibodyis encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 and/or comprisesan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 66.

In certain embodiments, a nucleic acid of the present disclosurecomprises a first polynucleotide sequence and a second polynucleotidesequence. The first and second polynucleotide sequence may be separatedby a linker. For example, in certain embodiments the antigen bindingdomain capable of binding PSCA and the antigen binding domain capable ofbinding PSMA are separated by a linker. In certain embodiments, thelinker is encoded by a polynucleotide sequence comprising the nucleicacid sequence set forth in SEQ ID NO: 53 or 54. A linker for use in thepresent disclosure allows for multiple proteins to be encoded by thesame nucleic acid sequence (e.g., a multicistronic or bicistronicsequence), which are translated as a polyprotein that is dissociatedinto separate protein components. For example, a linker for use in anucleic acid of the present disclosure comprising a PSCA CAR codingsequence and an PSMA CAR coding sequence, allows for the PSCA CAR andPSMA CAR to be translated as a polyprotein that is dissociated intoseparate CARs. In certain embodiments, the nucleic acid comprises from5′ to 3′ the first polynucleotide sequence, the linker, and the secondpolynucleotide sequence. In certain embodiments, the nucleic acidcomprises from 5′ to 3′ the second polynucleotide sequence, the linker,and the first polynucleotide sequence.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for an internal ribosome entry site (IRES). As used herein, “aninternal ribosome entry site” or “IRES” refers to an element thatpromotes direct internal ribosome entry to the initiation codon, such asATG, of a protein coding region, thereby leading to cap-independenttranslation of the gene. Various internal ribosome entry sites are knownto those of skill in the art, including, without limitation, IRESobtainable from viral or cellular mRNA sources, e.g., immunogloublinheavy-chain binding protein (BiP); vascular endothelial growth factor(VEGF); fibroblast growth factor 2; insulin-like growth factor;translational initiation factor eIF4G; yeast transcription factors TFIIDand HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus,aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloneymurine leukemia virus (MoMLV). Those of skill in the art would be ableto select the appropriate IRES for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for a self-cleaving peptide. As used herein, a “self-cleavingpeptide” or “2A peptide” refers to an oligopeptide that allow multipleproteins to be encoded as polyproteins, which dissociate into componentproteins upon translation. Use of the term “self-cleaving” is notintended to imply a proteolytic cleavage reaction. Various self-cleavingor 2A peptides are known to those of skill in the art, including,without limitation, those found in members of the Picornaviridae virusfamily, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis Avirus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1(PTV-1); and carioviruses such as Theilovirus and encephalomyocarditisviruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV arereferred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.Those of skill in the art would be able to select the appropriateself-cleaving peptide for use in the present invention.

In some embodiments, a linker further comprises a nucleic acid sequencethat encodes a furin cleavage site. Furin is a ubiquitously expressedprotease that resides in the trans-golgi and processes proteinprecursors before their secretion. Furin cleaves at the COOH— terminusof its consensus recognition sequence. Various furin consensusrecognition sequences (or “furin cleavage sites”) are known to those ofskill in the art, including, without limitation, Arg-X1-Lys-Arg (SEQ IDNO:143) or Arg-X1-Arg-Arg (SEQ ID NO:144), X2-Arg-X1-X3-Arg (SEQ IDNO:145) and Arg-X1-X1-Arg (SEQ ID NO:146), such as an Arg-Gln-Lys-Arg(SEQ ID NO:147), where X1 is any naturally occurring amino acid, X2 isLys or Arg, and X3 is Lys or Arg. Those of skill in the art would beable to select the appropriate Furin cleavage site for use in thepresent invention.

In some embodiments, the linker comprises a nucleic acid sequenceencoding a combination of a Furin cleavage site and a 2A peptide.Examples include, without limitation, a linker comprising a nucleic acidsequence encoding a Furin cleavage site and F2A, a linker comprising anucleic acid sequence encoding a Furin cleavage site and E2A, a linkercomprising a nucleic acid sequence encoding a Furin cleavage site andP2A, a linker comprising a nucleic acid sequence encoding a Furincleavage site and T2A. Those of skill in the art would be able to selectthe appropriate combination for use in the present invention. In suchembodiments, the linker may further comprise a spacer sequence betweenthe Furin cleavage site and the 2A peptide. In some embodiments, thelinker comprises a Furin cleavage site 5′ to a 2A peptide. In someembodiments, the linker comprises a 2A peptide 5′ to a Furin cleavagesite. Various spacer sequences are known in the art, including, withoutlimitation, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ IDNO:119) and (GGGS)n (SEQ ID NO:120), where n represents an integer of atleast 1. Exemplary spacer sequences can comprise amino acid sequencesincluding, without limitation, GGSG (SEQ ID NO:122), GGSGG (SEQ IDNO:123), GSGSG (SEQ ID NO:124), GSGGG (SEQ ID NO:125), GGGSG (SEQ IDNO:126), GSSSG (SEQ ID NO:127), and the like. Those of skill in the artwould be able to select the appropriate spacer sequence for use in thepresent invention.

In some embodiments, a nucleic acid of the present disclosure may beoperably linked to a transcriptional control element, e.g., a promoter,and enhancer, etc. Suitable promoter and enhancer elements are known tothose of skill in the art.

In certain embodiments, the nucleic acid encoding an exogenous CAR is inoperable linkage with a promoter. In certain embodiments, the promoteris a phosphoglycerate kinase-1 (PGK) promoter.

For expression in a bacterial cell, suitable promoters include, but arenot limited to, lad, lacZ, T3, T7, gpt, lambda P and trc. For expressionin a eukaryotic cell, suitable promoters include, but are not limitedto, light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. Suitable reversible promoters, including reversible induciblepromoters are known in the art. Such reversible promoters may beisolated and derived from many organisms, e.g., eukaryotes andprokaryotes. Modification of reversible promoters derived from a firstorganism for use in a second organism, e.g., a first prokaryote and asecond a eukaryote, a first eukaryote and a second a prokaryote, etc.,is well known in the art. Such reversible promoters, and systems basedon such reversible promoters but also comprising additional controlproteins, include, but are not limited to, alcohol regulated promoters(e.g., alcohol dehydrogenase I (alcA) gene promoter, promotersresponsive to alcohol transactivator proteins (A1cR), etc.),tetracycline regulated promoters, (e.g., promoter systems includingTetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g.,rat glucocorticoid receptor promoter systems, human estrogen receptorpromoter systems, retinoid promoter systems, thyroid promoter systems,ecdysone promoter systems, mifepristone promoter systems, etc.), metalregulated promoters (e.g., metallothionein promoter systems, etc.),pathogenesis-related regulated promoters (e.g., salicylic acid regulatedpromoters, ethylene regulated promoters, benzothiadiazole regulatedpromoters, etc.), temperature regulated promoters (e.g., heat shockinducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter,etc.), light regulated promoters, synthetic inducible promoters, and thelike.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an NcrI (p46) promoter; see, e.g., Eckelhart et al. Blood (2011)117:1565.

For expression in a yeast cell, a suitable promoter is a constitutivepromoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, aPYK1 promoter and the like; or a regulatable promoter such as a GALLpromoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDHpromoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use inPichia). Selection of the appropriate vector and promoter is well withinthe level of ordinary skill in the art. Suitable promoters for use inprokaryotic host cells include, but are not limited to, a bacteriophageT7 RNA polymerase promoter; a trp promoter; a lac operon promoter; ahybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybridpromoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tacpromoter, and the like; an araBAD promoter; in vivo regulated promoters,such as an ssaG promoter or a related promoter (see, e.g., U.S. PatentPublication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J.Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl.Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne etal. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan etal., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004)22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888-892); asigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBankAccession Nos. AX798980, AX798961, and AX798183); a stationary phasepromoter, e.g., a dps promoter, an spy promoter, and the like; apromoter derived from the pathogenicity island SPI-2 (see, e.g.,WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect.Immun. (2002) 70:1087-1096); an rpsM promoter (see, e.g., Valdivia andFalkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g.,Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U.(eds), Topics in Molecular and Structural Biology, Protein-Nucleic AcidInteraction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035);and the like. Suitable strong promoters for use in prokaryotes such asEscherichia coli include, but are not limited to Trc, Tac, T5, T7, andPLambda. Non-limiting examples of operators for use in bacterial hostcells include a lactose promoter operator (Lad repressor protein changesconformation when contacted with lactose, thereby preventing the Ladrepressor protein from binding to the operator), a tryptophan promoteroperator (when complexed with tryptophan, TrpR repressor protein has aconformation that binds the operator; in the absence of tryptophan, theTrpR repressor protein has a conformation that does not bind to theoperator), and a tac promoter operator (see, e.g., deBoer et al., Proc.Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

Other examples of suitable promoters include the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to a simian virus 40 (SV40) early promoter, a mouse mammarytumor virus (MMTV) or human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, a MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the EF-1 alpha promoter, as well as human gene promoterssuch as, but not limited to, an actin promoter, a myosin promoter, ahemoglobin promoter, and a creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In some embodiments, the locus or construct or transgene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99,the disclosure of which is incorporated herein by reference). Anysuitable combination of recombinase, endonuclease, ligase, recombinationsites, etc. known to the art may be used in generating an irreversiblyswitchable promoter. Methods, mechanisms, and requirements forperforming site-specific recombination, described elsewhere herein, finduse in generating irreversibly switched promoters and are well known inthe art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006)567-605; and Tropp, Molecular Biology (2012) (Jones & BartlettPublishers, Sudbury, Mass.), the disclosures of which are incorporatedherein by reference.

In some embodiments, a nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding a CAR inducible expressioncassette. In one embodiment, the CAR inducible expression cassette isfor the production of a transgenic polypeptide product that is releasedupon CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol.Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5):535-544. In some embodiments, a nucleic acid of the present disclosurefurther comprises a nucleic acid sequence encoding a cytokine operablylinked to a T-cell activation responsive promoter. In some embodiments,the cytokine operably linked to a T-cell activation responsive promoteris present on a separate nucleic acid sequence. In one embodiment, thecytokine is IL-12.

A nucleic acid of the present disclosure may be present within anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.Suitable expression vectors include, e.g., plasmids, viral vectors, andthe like. Large numbers of suitable vectors and promoters are known tothose of skill in the art; many are commercially available forgenerating a subject recombinant construct. The following vectors areprovided by way of example, and should not be construed in anyway aslimiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest.Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther.(1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995)92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. GeneTher. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum.Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus(see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); aretroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus); and thelike.

Additional expression vectors suitable for use are, e.g., withoutlimitation, a lentivirus vector, a gamma retrovirus vector, a foamyvirus vector, an adeno-associated virus vector, an adenovirus vector, apox virus vector, a herpes virus vector, an engineered hybrid virusvector, a transposon mediated vector, and the like. Viral vectortechnology is well known in the art and is described, for example, inSambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes1-4, Cold Spring Harbor Press, NY), and in other virology and molecularbiology manuals. Viruses, which are useful as vectors include, but arenot limited to, retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses.

In general, a suitable vector contains an origin of replicationfunctional in at least one organism, a promoter sequence, convenientrestriction endonuclease sites, and one or more selectable markers,(e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

In some embodiments, an expression vector (e.g., a lentiviral vector)may be used to introduce the CAR into an immune cell or precursorthereof (e.g., a T cell). Accordingly, an expression vector (e.g., alentiviral vector) of the present invention may comprise a nucleic acidencoding for a CAR. In some embodiments, the expression vector (e.g.,lentiviral vector) will comprise additional elements that will aid inthe functional expression of the CAR encoded therein. In someembodiments, an expression vector comprising a nucleic acid encoding fora CAR further comprises a mammalian promoter. In one embodiment, thevector further comprises an elongation-factor-1-alpha promoter (EF-1αpromoter). Use of an EF-1α promoter may increase the efficiency inexpression of downstream transgenes (e.g., a CAR encoding nucleic acidsequence). Physiologic promoters (e.g., an EF-1α promoter) may be lesslikely to induce integration mediated genotoxicity, and may abrogate theability of the retroviral vector to transform stem cells. Otherphysiological promoters suitable for use in a vector (e.g., lentiviralvector) are known to those of skill in the art and may be incorporatedinto a vector of the present invention. In some embodiments, the vector(e.g., lentiviral vector) further comprises a non-requisite cis actingsequence that may improve titers and gene expression. One non-limitingexample of a non-requisite cis acting sequence is the central polypurinetract and central termination sequence (cPPT/CTS) which is important forefficient reverse transcription and nuclear import. Other non-requisitecis acting sequences are known to those of skill in the art and may beincorporated into a vector (e.g., lentiviral vector) of the presentinvention. In some embodiments, the vector further comprises aposttranscriptional regulatory element. Posttranscriptional regulatoryelements may improve RNA translation, improve transgene expression andstabilize RNA transcripts. One example of a posttranscriptionalregulatory element is the woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE). Accordingly, in some embodiments a vector forthe present invention further comprises a WPRE sequence. Variousposttranscriptional regulator elements are known to those of skill inthe art and may be incorporated into a vector (e.g., lentiviral vector)of the present invention. A vector of the present invention may furthercomprise additional elements such as a rev response element (RRE) forRNA transport, packaging sequences, and 5′ and 3′ long terminal repeats(LTRs). The term “long terminal repeat” or “LTR” refers to domains ofbase pairs located at the ends of retroviral DNAs which comprise U3, Rand U5 regions. LTRs generally provide functions required for theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. In oneembodiment, a vector (e.g., lentiviral vector) of the present inventionincludes a 3′ U3 deleted LTR. Accordingly, a vector (e.g., lentiviralvector) of the present invention may comprise any combination of theelements described herein to enhance the efficiency of functionalexpression of transgenes. For example, a vector (e.g., lentiviralvector) of the present invention may comprise a WPRE sequence, cPPTsequence, RRE sequence, 5′LTR, 3′ U3 deleted LTR′ in addition to anucleic acid encoding for a CAR.

Vectors of the present invention may be self-inactivating vectors. Asused herein, the term “self-inactivating vector” refers to vectors inwhich the 3′ LTR enhancer promoter region (U3 region) has been modified(e.g., by deletion or substitution). A self-inactivating vector mayprevent viral transcription beyond the first round of viral replication.Consequently, a self-inactivating vector may be capable of infecting andthen integrating into a host genome (e.g., a mammalian genome) onlyonce, and cannot be passed further. Accordingly, self-inactivatingvectors may greatly reduce the risk of creating a replication-competentvirus.

In some embodiments, a nucleic acid of the present invention may be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known to those of skill in the art; any known method can be used tosynthesize RNA comprising a sequence encoding a CAR of the presentdisclosure. Methods for introducing RNA into a host cell are known inthe art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. IntroducingRNA comprising a nucleotide sequence encoding a CAR of the presentdisclosure into a host cell can be carried out in vitro, ex vivo or invivo. For example, a host cell (e.g., an NK cell, a cytotoxic Tlymphocyte, etc.) can be electroporated in vitro or ex vivo with RNAcomprising a nucleotide sequence encoding a CAR of the presentdisclosure.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell may also containeither a selectable marker gene or a reporter gene, or both, tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In some embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, without limitation, antibiotic-resistancegenes.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assessed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include, without limitation, genesencoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescentprotein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

In some embodiments, a nucleic acid of the present disclosure isprovided for the production of a CAR as described herein, e.g., in amammalian cell. In some embodiments, a nucleic acid of the presentdisclosure provides for amplification of the CAR-encoding nucleic acid.

D. Modified Immune Cells

The present invention provides modified immune cells or precursorsthereof (e.g., T cells) comprising chimeric antigen receptors (CARs)capable of binding PSCA (e.g. human PSCA). Also provided are modifiedimmune cells or precursors thereof comprising bispecific CARs (e.g. PSCA& PSMA), PSCA CARs with a dominant negative receptor (e.g., TGFbRDN),PSCA CARs with a switch receptor (e.g., PD1/CD28 or TGFbR/IL12R), andPSCA CARs in combination with bispecific antibodies (e.g., PD-L1/CD28).The invention also includes modified immune cells or precursors thereofcomprising any of the nucleic acids disclosed herein or any of thevectors disclosed herein.

In one aspect, the invention includes a modified immune cell orprecursor cell thereof, comprising a CAR comprising an antigen-bindingdomain capable of binding PSCA, a transmembrane domain, and anintracellular domain.

In certain exemplary embodiments, the antigen binding domain comprisesthree heavy chain complementarity determining regions (HCDRs), whereinHCDR1 comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2comprises the amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), andHCDR3 comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6).

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof, comprising a chimeric antigen receptor(CAR) capable of binding prostate stem cell antigen (PSCA), comprisingan antigen binding domain, a transmembrane domain, and an intracellulardomain, wherein the antigen-binding domain comprises: a heavy chainvariable region comprising an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7; and alight chain variable region comprising an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof, comprising a chimeric antigen receptor(CAR) capable of binding prostate stem cell antigen (PSCA), comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 9.

In certain exemplary embodiments, the transmembrane domain comprises atransmembrane domain of CD8 alpha.

In certain exemplary embodiments, the intracellular domain comprises acostimulatory signaling domain and an intracellular signaling domain. Incertain exemplary embodiments, the costimulatory signaling domaincomprises a costimulatory domain of 4-1BB. In certain exemplaryembodiments, the costimulatory signaling domain comprises acostimulatory domain of CD28. In certain exemplary embodiments, thecostimulatory signaling domain comprises a costimulatory domain of ICOS.In certain exemplary embodiments, the costimulatory signaling domaincomprises a costimulatory domain of ICOS(YMNM). In certain exemplaryembodiments, the intracellular signaling domain comprises anintracellular domain of CD3 or a variant thereof.

In another aspect, the instant disclosure provides a modified immunecell or precursor cell thereof, comprising a chimeric antigen receptor(CAR) capable of binding prostate stem cell antigen (PSCA), comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 21, 23, 25 or 27.

In certain exemplary embodiments, the modified cell further comprises aPSMA-CAR, wherein the PSMA-CAR comprises an antigen-binding domain, atransmembrane domain, and an intracellular domain.

In one aspect, the invention includes a modified immune cell orprecursor cell thereof comprising a bispecific chimeric antigen receptor(CAR) comprising an extracellular domain comprising an antigen-bindingdomain capable of binding prostate stem cell antigen (PSCA) and anantigen-binding domain capable of binding prostate specific membraneantigen (PSMA).

In certain embodiments, the extracellular domain of the bispecific CARcomprises an antigen binding domain capable of binding PSCA comprising:a first heavy chain variable region that comprises three heavy chaincomplementarity determining regions (HCDRs), wherein HCDR1 comprises theamino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprises the amino acidsequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3 comprises the aminoacid sequence TGGF (SEQ ID NO: 3); and a first light chain variableregion that comprises three light chain complementarity determiningregions (LCDRs), wherein LCDR1 comprises the amino acid sequenceSASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acid sequenceDTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequenceQQWSSSPFT (SEQ ID NO: 6). The extracellular domain also comprises anantigen binding domain capable of binding PSMA comprising: a secondheavy chain variable region that comprises three heavy chaincomplementarity determining regions (HCDRs), wherein HCDR1 comprises theamino acid sequence SNWIG (SEQ ID NO: 28), HCDR2 comprises the aminoacid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 29), and HCDR3 comprises theamino acid sequence QTGFLWSFDL (SEQ ID NO: 30); and a second light chainvariable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence RASQDISSALA (SEQ ID NO: 31), LCDR2 comprises the amino acidsequence DASSLES (SEQ ID NO: 32), and LCDR3 comprises the amino acidsequence QQFNSYPLT (SEQ ID NO: 33).

In certain embodiments, the extracellular domain comprises an antigenbinding domain capable of binding PSCA comprising: a first heavy chainvariable region comprising an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7; and afirst light chain variable region comprising an amino acid sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQID NO: 8 and/or the antigen binding domain capable of binding PSMAcomprises: a second heavy chain variable region comprising an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 34; and a second light chain variable regioncomprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 35.

In certain embodiments, the extracellular domain of the bispecific CARcomprises an antigen binding domain capable of binding PSCA comprising:a first heavy chain variable region that comprises three heavy chaincomplementarity determining regions (HCDRs), wherein HCDR1 comprises theamino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprises the amino acidsequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3 comprises the aminoacid sequence TGGF (SEQ ID NO: 3); and a first light chain variableregion that comprises three light chain complementarity determiningregions (LCDRs), wherein LCDR1 comprises the amino acid sequenceSASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acid sequenceDTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequenceQQWSSSPFT (SEQ ID NO: 6) and/or the antigen binding domain capable ofbinding PSMA comprises: a second heavy chain variable region thatcomprises three heavy chain complementarity determining regions (HCDRs),wherein HCDR1 comprises the amino acid sequence EYTIH (SEQ ID NO: 68),HCDR2 comprises the amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO:69), and HCDR3 comprises the amino acid sequence GWNFDY (SEQ ID NO: 70);and a second light chain variable region that comprises three lightchain complementarity determining regions (LCDRs), wherein LCDR1comprises the amino acid sequence KASQDVGTAVD (SEQ ID NO: 71), LCDR2comprises the amino acid sequence WASTRHT (SEQ ID NO: 72), and LCDR3comprises the amino acid sequence QQYNSYPLT (SEQ ID NO: 73).

In certain embodiments, the extracellular domain of the bispecific CARcomprises an antigen binding domain capable of binding PSCA comprising:a first heavy chain variable region comprising an amino acid sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQID NO: 7; and a first light chain variable region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 8 and/or the antigen binding domain capable ofbinding PSMA comprises: a second heavy chain variable region comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 75; and a second light chain variableregion comprising an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77.

In certain exemplary embodiments, the bispecific CAR is capable ofbinding human PSCA. In certain exemplary embodiments, the bispecific CARis capable of binding human PSMA. In certain exemplary embodiments, thebispecific CAR is capable of binding human PSCA and human PSMA.

In another aspect, the invention includes a modified immune cell orprecursor cell thereof, comprising a first CAR comprising a firstantigen binding domain capable of binding prostate stem cell antigen(PSCA) and a second CAR comprising a second antigen binding domaincapable of binding prostate specific membrane antigen (PSMA), whereinthe first and the second CAR each comprise a transmembrane domain and anintracellular domain.

In certain exemplary embodiments, the first antigen binding domaincomprises: a first heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a first lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6). The second antigen binding domaincomprises: a second heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence SNWIG (SEQ ID NO: 28), HCDR2 comprisesthe amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 29), and HCDR3comprises the amino acid sequence QTGFLWSFDL (SEQ ID NO: 30); and asecond light chain variable region that comprises three light chaincomplementarity determining regions (LCDRs), wherein LCDR1 comprises theamino acid sequence RASQDISSALA (SEQ ID NO: 31), LCDR2 comprises theamino acid sequence DASSLES (SEQ ID NO: 32), and LCDR3 comprises theamino acid sequence QQFNSYPLT (SEQ ID NO: 33).

In certain exemplary embodiments, the first heavy chain variable regioncomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 7; and/or the first lightchain variable region comprises an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8;and/or the second heavy chain variable region comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 34; and/or the second light chain variableregion comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35.

In certain exemplary embodiments, the first antigen binding domaincomprises: a first heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKFQG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and a first lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6). The second antigen binding domaincomprises: a second heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence EYTIH (SEQ ID NO: 68), HCDR2 comprisesthe amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO: 69), and HCDR3comprises the amino acid sequence GWNFDY (SEQ ID NO: 70); and a secondlight chain variable region that comprises three light chaincomplementarity determining regions (LCDRs), wherein LCDR1 comprises theamino acid sequence KASQDVGTAVD (SEQ ID NO: 71), LCDR2 comprises theamino acid sequence WASTRHT (SEQ ID NO: 72), and LCDR3 comprises theamino acid sequence QQYNSYPLT (SEQ ID NO: 73).

In certain exemplary embodiments, the first heavy chain variable regioncomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 7; and/or the first lightchain variable region comprises an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8;and/or the second heavy chain variable region comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 75; and/or the second light chain variableregion comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77.

In certain exemplary embodiments, the first CAR and the second CAR eachcomprise a transmembrane domain selected from the group consisting of anartificial hydrophobic sequence, and a transmembrane domain of a type Itransmembrane protein, an alpha, beta, or zeta chain of a T cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS, and CD154, ora transmembrane domain derived from a killer immunoglobulin-likereceptor (KIR). In certain exemplary embodiments, the first CAR and thesecond CAR each comprise a transmembrane domain of CD8 alpha.

In certain exemplary embodiments, the intracellular domain of the firstCAR and the second CAR each comprise a costimulatory signaling domainand an intracellular signaling domain.

In certain exemplary embodiments, the intracellular domain of the firstCAR and the second CAR each comprise a costimulatory domain of a proteinselected from the group consisting of proteins in the TNFR superfamily,CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10,DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30,CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or anintracellular domain derived from a killer immunoglobulin-like receptor(KIR). In certain exemplary embodiments, the intracellular domain of thefirst CAR and the second CAR each comprise a costimulatory domain of4-1BB.

In certain exemplary embodiments, the intracellular signaling domain ofthe first CAR and the second CAR each comprise an intracellular domainselected from the group consisting of cytoplasmic signaling domains of ahuman CD3 zeta chain (CD3), FcγRIII, FcsRI, a cytoplasmic tail of an Fcreceptor, an immunoreceptor tyrosine-based activation motif (ITAM)bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta,CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.In certain exemplary embodiments, the intracellular signaling domain ofthe first CAR and the second CAR each comprise an intracellular domainof CD3 or a variant thereof.

In certain exemplary embodiments, the first CAR comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 21 and/or the second CAR comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 67, 87, 89, 91, 93, 95, or 97.

In certain embodiments, the modified cell further comprises a dominantnegative receptor. In certain embodiments, the dominant negativereceptor is a truncated variant of a wild-type protein associated with anegative signal. In certain exemplary embodiments, the truncated variantof a wild-type protein associated with a negative signal comprises anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 56.

In certain embodiments, the modified cell further comprises a switchreceptor. In certain embodiments, the switch receptor comprises a firstdomain derived from a first switch polypeptide that is associated with anegative signal; and a second domain derived from a second switchpolypeptide that is associated with a positive signal. In certainexemplary embodiments, the switch receptor comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 58, 60, or 62.

Accordingly, in certain embodiments, the invention includes a modifiedimmune cell or precursor cell thereof, comprising a CAR comprising anantigen-binding domain capable of binding PSCA, a transmembrane domain,and an intracellular domain, wherein the cell further comprises adominant negative receptor (e.g., TGFbRDN).

Accordingly, in certain embodiments, the invention includes a modifiedimmune cell or precursor cell thereof, comprising a CAR comprising anantigen-binding domain capable of binding PSCA, a transmembrane domain,and an intracellular domain, wherein the cell further comprises a switchreceptor (e.g., a PD1/CD28 switch receptor).

Also provided is a modified immune cell or precursor cell thereof,comprising a CAR capable of binding prostate stem cell antigen(PSCA-CAR), and a bispecific antibody, wherein the CAR comprises anantigen-binding domain, a transmembrane domain, and an intracellulardomain, and wherein the modified cell secretes the bispecific antibody.

In certain embodiments, the modified cell further comprises a abispecific antibody, wherein the cell secretes the bispecific antibody.In certain embodiments, the bispecific antibody comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 64 or 66.

In certain embodiments, the modified cell is a modified immune cell. Incertain embodiments, the modified cell is a modified T cell. In certainembodiments, the the modified cell is an autologous cell. In certainembodiments, the modified cell is an autologous cell obtained from ahuman subject.

E. Sources of Immune Cells

In certain embodiments, a source of immune cells (e.g. T cells) isobtained from a subject for ex vivo manipulation. Sources of immunecells for ex vivo manipulation may also include, e.g., autologous orheterologous donor blood, cord blood, or bone marrow. For example thesource of immune cells may be from the subject to be treated with themodified immune cells of the invention, e.g., the subject's blood, thesubject's cord blood, or the subject's bone marrow. Non-limitingexamples of subjects include humans, dogs, cats, mice, rats, andtransgenic species thereof. Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood,peripheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cellsare cells of the immune system, such as cells of the innate or adaptiveimmunity, e.g., myeloid or lymphoid cells, including lymphocytes,typically T cells and/or NK cells. Other exemplary cells include stemcells, such as multipotent and pluripotent stem cells, including inducedpluripotent stem cells (iPSCs). In some aspects, the cells are humancells. With reference to the subject to be treated, the cells may beallogeneic and/or autologous. The cells typically are primary cells,such as those isolated directly from a subject and/or isolated from asubject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell(e.g., a CD8+ naive T cell, central memory T cell, or effector memory Tcell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatoryT cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell ahematopoietic stem cell, a natural killer cell (NK cell) or a dendriticcell. In some embodiments, the cells are monocytes or granulocytes,e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, and/or basophils. In an embodiment, the target cellis an induced pluripotent stem (iPS) cell or a cell derived from an iPScell, e.g., an iPS cell generated from a subject, manipulated to alter(e.g., induce a mutation in) or manipulate the expression of one or moretarget genes, and differentiated into, e.g., a T cell, e.g., a CD8+ Tcell (e.g., a CD8+ naive T cell, central memory T cell, or effectormemory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoidprogenitor cell or a hematopoietic stem cell.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. Among the sub-types and subpopulations of Tcells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells,effector T cells (TEFF), memory T cells and sub-types thereof, such asstem cell memory T (TSCM), central memory T (TCM), effector memory T(TEM), or terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MATT)cells, naturally occurring and adaptive regulatory T (Treg) cells,helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9cells, TH22 cells, follicular helper T cells, alpha/beta T cells, anddelta/gamma T cells. In certain embodiments, any number of T cell linesavailable in the art, may be used.

In some embodiments, the methods include isolating immune cells from thesubject, preparing, processing, culturing, and/or engineering them. Insome embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for engineering asdescribed may be isolated from a sample, such as a biological sample,e.g., one obtained from or derived from a subject. In some embodiments,the subject from which the cell is isolated is one having the disease orcondition or in need of a cell therapy or to which cell therapy will beadministered. The subject in some embodiments is a human in need of aparticular therapeutic intervention, such as the adoptive cell therapyfor which cells are being isolated, processed, and/or engineered.Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig. Insome embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets. In some embodiments, the blood cellscollected from the subject are washed, e.g., to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In some embodiments, the cells are washedwith phosphate buffered saline (PBS). In some aspects, a washing step isaccomplished by tangential flow filtration (TFF) according to themanufacturer's instructions. In some embodiments, the cells areresuspended in a variety of biocompatible buffers after washing. Incertain embodiments, components of a blood cell sample are removed andthe cells directly resuspended in culture media. In some embodiments,the methods include density-based cell separation methods, such as thepreparation of white blood cells from peripheral blood by lysing the redblood cells and centrifugation through a Percoll or Ficoll gradient.

In one embodiment, immune are obtained cells from the circulating bloodof an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media, such as phosphate buffered saline (PBS) orwash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population. The separation need not result in 100%enrichment or removal of a particular cell population or cellsexpressing a particular marker. For example, positive selection of orenrichment for cells of a particular type, such as those expressing amarker, refers to increasing the number or percentage of such cells, butneed not result in a complete absence of cells not expressing themarker. Likewise, negative selection, removal, or depletion of cells ofa particular type, such as those expressing a marker, refers todecreasing the number or percentage of such cells, but need not resultin a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

In some embodiments, one or more of the T cell populations is enrichedfor or depleted of cells that are positive for (marker+) or express highlevels (marker^(high)) of one or more particular markers, such assurface markers, or that are negative for (marker −) or expressrelatively low levels (marker^(low)) of one or more markers. Forexample, in some aspects, specific subpopulations of T cells, such ascells positive or expressing high levels of one or more surface markers,e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/orCD45RO+ T cells, are isolated by positive or negative selectiontechniques. In some cases, such markers are those that are absent orexpressed at relatively low levels on certain populations of T cells(such as non-memory cells) but are present or expressed at relativelyhigher levels on certain other populations of T cells (such as memorycells). In one embodiment, the cells (such as the CD8+ cells or the Tcells, e.g., CD3+ cells) are enriched for (i.e., positively selectedfor) cells that are positive or expressing high surface levels ofCD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of(e.g., negatively selected for) cells that are positive for or expresshigh surface levels of CD45RA. In some embodiments, cells are enrichedfor or depleted of cells positive or expressing high surface levels ofCD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+T cells are enriched for cells positive for CD45R0 (or negative forCD45RA) and for CD62L. For example, CD3+, CD28+ T cells can bepositively selected using CD3/CD28 conjugated magnetic beads (e.g.,DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD 14. In some aspects, aCD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations. In someembodiments, CD8+ cells are further enriched for or depleted of naive,central memory, effector memory, and/or central memory stem cells, suchas by positive or negative selection based on surface antigensassociated with the respective subpopulation. In some embodiments,enrichment for central memory T (TCM) cells is carried out to increaseefficacy, such as to improve long-term survival, expansion, and/orengraftment following administration, which in some aspects isparticularly robust in such sub-populations. In some embodiments,combining TCM-enriched CD8+ T cells and CD4+ T cells further enhancesefficacy.

In some embodiments, memory T cells are present in both CD62L+ andCD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can beenriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, suchas using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+T cell population and a CD8+ T cell sub-population, e.g., asub-population enriched for central memory (TCM) cells. In someembodiments, the enrichment for central memory T (TCM) cells is based onpositive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3,and/or CD 127; in some aspects, it is based on negative selection forcells expressing or highly expressing CD45RA and/or granzyme B. In someaspects, isolation of a CD8+ population enriched for TCM cells iscarried out by depletion of cells expressing CD4, CD 14, CD45RA, andpositive selection or enrichment for cells expressing CD62L. In oneaspect, enrichment for central memory T (TCM) cells is carried outstarting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD 14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ cell population or subpopulation, also is used to generate theCD4+ cell population or sub-population, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

CD4+T helper cells are sorted into naive, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+ cells are CD62L+ andCD45R0+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO.In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor. The conditions caninclude one or more of particular media, temperature, oxygen content,carbon dioxide content, time, agents, e.g., nutrients, amino acids,antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors, and any other agents designed to activate the cells.In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from an umbilical cord. In any event, a specificsubpopulation of T cells can be further isolated by positive or negativeselection techniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19, and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4⁺ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° C. per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

In one embodiment, the T cell is comprised within a population of cellssuch as peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of T cells.In yet another embodiment, purified T cells comprise the population of Tcells.

In certain embodiments, T regulatory cells (Tregs) can be isolated froma sample. The sample can include, but is not limited to, umbilical cordblood or peripheral blood. In certain embodiments, the Tregs areisolated by flow-cytometry sorting. The sample can be enriched for Tregsprior to isolation by any means known in the art. The isolated Tregs canbe cryopreserved, and/or expanded prior to use. Methods for isolatingTregs are described in U.S. Pat. Nos. 7,754,482, 8,722,400, and9,555,105, and U.S. patent application Ser. No. 13/639,927, contents ofwhich are incorporated herein in their entirety.

F. Methods of Treatment

The modified immune cells (e.g., T cells) described herein may beincluded in a composition for immunotherapy. The composition may includea pharmaceutical composition and further include a pharmaceuticallyacceptable carrier. A therapeutically effective amount of thepharmaceutical composition comprising the modified T cells may beadministered.

In one aspect, the invention includes a method of treating a disease orcondition in a subject comprising administering to a subject in needthereof an effective amount of a modified cell (e.g. T cell) of thepresent invention. In another aspect, the invention includes a method oftreating a disease or condition in a subject comprising administering toa subject in need thereof a pharmaceutical composition comprising aneffective amount of a modified cell (e.g. T cell) of the presentinvention. In another aspect, the invention includes a method foradoptive cell transfer therapy comprising administering to a subject inneed thereof an effective amount of a modified cell (e.g. T cell) of thepresent invention.

Methods for administration of immune cells for adoptive cell therapy areknown and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, the cell therapy, e.g., adoptive T celltherapy is carried out by autologous transfer, in which the cells areisolated and/or otherwise prepared from the subject who is to receivethe cell therapy, or from a sample derived from such a subject. Thus, insome aspects, the cells are derived from a subject, e.g., patient, inneed of a treatment and the cells, following isolation and processingare administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In some embodiments, the subject has been treated with a therapeuticagent targeting the disease or condition, e.g. the tumor, prior toadministration of the cells or composition containing the cells. In someaspects, the subject is refractory or non-responsive to the othertherapeutic agent. In some embodiments, the subject has persistent orrelapsed disease, e.g., following treatment with another therapeuticintervention, including chemotherapy, radiation, and/or hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT. In someembodiments, the administration effectively treats the subject despitethe subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some aspects, the subject is initially responsive to the therapeuticagent, but exhibits a relapse of the disease or condition over time. Insome embodiments, the subject has not relapsed. In some suchembodiments, the subject is determined to be at risk for relapse, suchas at a high risk of relapse, and thus the cells are administeredprophylactically, e.g., to reduce the likelihood of or prevent relapse.In some aspects, the subject has not received prior treatment withanother therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapeutic intervention,including chemotherapy, radiation, and/or hematopoietic stem celltransplantation (HSCT), e.g., allogenic HSCT. In some embodiments, theadministration effectively treats the subject despite the subject havingbecome resistant to another therapy.

The modified immune cells of the present invention can be administeredto an animal, preferably a mammal, even more preferably a human, totreat a cancer. In addition, the cells of the present invention can beused for the treatment of any condition related to a cancer, especiallya cell-mediated immune response against a tumor cell(s), where it isdesirable to treat or alleviate the disease. The types of cancers to betreated with the modified cells or pharmaceutical compositions of theinvention include, carcinoma, blastoma, and sarcoma, and certainleukemia or lymphoid malignancies, benign and malignant tumors, andmalignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplarycancers include but are not limited breast cancer, prostate cancer,ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, thyroid cancer, and the like. The cancers may benon-solid tumors (such as hematological tumors) or solid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included. In oneembodiment, the cancer is a solid tumor or a hematological tumor. In oneembodiment, the cancer is a carcinoma. In one embodiment, the cancer isa sarcoma. In one embodiment, the cancer is a leukemia. In oneembodiment the cancer is a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases). In certain embodiments, the cancer is an astrocytoma. Incertain embodiments, the cancer is a high-grade astrocytoma. In certainembodiments, the cancer is prostate cancer. In certain embodiments, thecancer is metastatic castrate resistant prostate cancer.

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a myeloma, or a condition related tomyeloma. Examples of myeloma or conditions related thereto include,without limitation, light chain myeloma, non-secretory myeloma,monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma(e.g., solitary, multiple solitary, extramedullary plasmacytoma),amyloidosis, and multiple myeloma. In one embodiment, a method of thepresent disclosure is used to treat multiple myeloma. In one embodiment,a method of the present disclosure is used to treat refractory myeloma.In one embodiment, a method of the present disclosure is used to treatrelapsed myeloma.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a melanoma, or a condition related tomelanoma. Examples of melanoma or conditions related thereto include,without limitation, superficial spreading melanoma, nodular melanoma,lentigo maligna melanoma, acral lentiginous melanoma, amelanoticmelanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina,rectum melanoma). In one embodiment, a method of the present disclosureis used to treat cutaneous melanoma. In one embodiment, a method of thepresent disclosure is used to treat refractory melanoma. In oneembodiment, a method of the present disclosure is used to treat relapsedmelanoma.

In yet other exemplary embodiments, the modified immune cells of theinvention are used to treat a sarcoma, or a condition related tosarcoma. Examples of sarcoma or conditions related thereto include,without limitation, angiosarcoma, chondrosarcoma, Ewing's sarcoma,fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma,liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma,pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In oneembodiment, a method of the present disclosure is used to treat synovialsarcoma. In one embodiment, a method of the present disclosure is usedto treat liposarcoma such as myxoid/round cell liposarcoma,differentiated/dedifferentiated liposarcoma, and pleomorphicliposarcoma. In one embodiment, a method of the present disclosure isused to treat myxoid/round cell liposarcoma. In one embodiment, a methodof the present disclosure is used to treat a refractory sarcoma. In oneembodiment, a method of the present disclosure is used to treat arelapsed sarcoma.

The cells of the invention to be administered may be autologous, withrespect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out inany convenient manner known to those of skill in the art. The cells ofthe present invention may be administered to a subject by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymphnode, an organ, a tumor, and the like.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4+ to CD8+ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺and CD4⁺ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsubtype, or minimum number of cells of the population or sub-type perunit of body weight. Thus, in some embodiments, the dosage is based on adesired fixed dose of total cells and a desired ratio, and/or based on adesired fixed dose of one or more, e.g., each, of the individualsub-types or sub-populations. Thus, in some embodiments, the dosage isbased on a desired fixed or minimum dose of T cells and a desired ratioof CD4⁺ to CD8⁺ cells, and/or is based on a desired fixed or minimumdose of CD4⁺ and/or CD8⁺ cells.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells, such as, e.g., 1 million toabout 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), such asabout 10 million to about 100 billion cells (e.g., about 20 millioncells, about 30 million cells, about 40 million cells, about 60 millioncells, about 70 million cells, about 80 million cells, about millioncells, about 10 billion cells, about 25 billion cells, about 50 billioncells, about 75 billion cells, about 90 billion cells, or a rangedefined by any two of the foregoing values), and in some cases about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about 1×10⁵cells/kg to about 1×10¹¹ cells/kg 10⁴ and at or about 10¹¹cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ cells/kgbody weight, for example, at or about 1×10⁵ cells/kg, 1.5×10⁵ cells/kg,2×10⁵ cells/kg, or 1×10⁶ cells/kg body weight. For example, in someembodiments, the cells are administered at, or within a certain range oferror of, between at or about 10⁴ and at or about 10⁹ T cells/kilograms(kg) body weight, such as between 10⁵ and 10⁶ T cells/kg body weight,for example, at or about 1×10⁵ T cells/kg, 1.5×10⁵ T cells/kg, 2×10⁵ Tcells/kg, or 1×10⁶ T cells/kg body weight. In other exemplaryembodiments, a suitable dosage range of modified cells for use in amethod of the present disclosure includes, without limitation, fromabout 1×10⁵ cells/kg to about 1×10⁶ cells/kg, from about 1×10⁶ cells/kgto about 1×10⁷ cells/kg, from about 1×10⁷ cells/kg about 1×10⁸ cells/kg,from about 1×10⁸ cells/kg about 1×10⁹ cells/kg, from about 1×10⁹cells/kg about 1×10¹⁰ cells/kg, from about 1×10¹⁰ cells/kg about 1×10¹¹cells/kg. In an exemplary embodiment, a suitable dosage for use in amethod of the present disclosure is about 1×10⁸ cells/kg. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 1×10⁷ cells/kg. In other embodiments, asuitable dosage is from about 1×10⁷ total cells to about 5×10⁷ totalcells. In some embodiments, a suitable dosage is from about 1×10⁸ totalcells to about 5×10⁸ total cells. In some embodiments, a suitable dosageis from about 1.4×10⁷ total cells to about 1.1×10⁹ total cells. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 7×10⁹ total cells.

In some embodiments, the cells are administered at or within a certainrange of error of between at or about 10⁴ and at or about 10⁹ CD4⁺and/or CD8+ cells/kilograms (kg) body weight, such as between 10⁵ and10⁶ CD4⁺ and/or CD8⁺ cells/kg body weight, for example, at or about1×10⁵ CD4⁺ and/or CD8⁺ cells/kg, 1.5×10⁵ CD4⁺ and/or CD8⁺ cells/kg,2×10⁵ CD4⁺ and/or CD8⁺ cells/kg, or 1×10⁶ CD4⁺ and/or CD8⁺ cells/kg bodyweight. In some embodiments, the cells are administered at or within acertain range of error of, greater than, and/or at least about 1×10⁶,about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ CD4⁺ cells,and/or at least about 1×10⁶, about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶,or about 9×10⁶ CD8⁺ cells, and/or at least about 1×10⁶, about 2.5×10⁶,about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ T cells. In some embodiments,the cells are administered at or within a certain range of error ofbetween about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ T cells,between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD4⁺ cells,and/or between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD8⁺cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios,for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

In some embodiments, a dose of modified cells is administered to asubject in need thereof, in a single dose or multiple doses. In someembodiments, a dose of modified cells is administered in multiple doses,e.g., once a week or every 7 days, once every 2 weeks or every 14 days,once every 3 weeks or every 21 days, once every 4 weeks or every 28days. In an exemplary embodiment, a single dose of modified cells isadministered to a subject in need thereof. In an exemplary embodiment, asingle dose of modified cells is administered to a subject in needthereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence. In some embodiments,the methods comprise administration of a chemotherapeutic agent.

In certain embodiments, the modified cells of the invention (e.g., amodified cell comprising a CAR) may be administered to a subject incombination with an inhibitor of an immune checkpoint. Examples ofimmune checkpoints include but are not limited to CTLA-4, PD-1, andTIM-3. Antibodies may be used to inhibit an immune checkpoint (e.g., ananti-PD1, anti-CTLA-4, or anti-TIM-3 antibody). For example, themodified cell may be administered in combination with an antibody orantibody fragment targeting, for example, PD-1 (programmed death 1protein). Examples of anti-PD-1 antibodies include, but are not limitedto, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known asMK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or anantigen-binding fragment thereof. In certain embodiments, the modifiedcell may be administered in combination with an anti-PD-L1 antibody orantigen-binding fragment thereof. Examples of anti-PD-L1 antibodiesinclude, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®,Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certainembodiments, the modified cell may be administered in combination withan anti-CTLA-4 antibody or antigen-binding fragment thereof. An exampleof an anti-CTLA-4 antibody includes, but is not limited to, Ipilimumab(trade name Yervoy). Other types of immune checkpoint modulators mayalso be used including, but not limited to, small molecules, siRNA,miRNA, and CRISPR systems. Immune checkpoint modulators may beadministered before, after, or concurrently with the modified cellcomprising the CAR. In certain embodiments, combination treatmentcomprising an immune checkpoint modulator may increase the therapeuticefficacy of a therapy comprising a modified cell of the presentinvention.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable method knownin the art, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al. J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load.

In certain embodiments, the subject is provided a secondary treatment.Secondary treatments include but are not limited to chemotherapy,radiation, surgery, and medications.

In some embodiments, the subject can be administered a conditioningtherapy prior to CAR T cell therapy. In some embodiments, theconditioning therapy comprises administering an effective amount ofcyclophosphamide to the subject. In some embodiments, the conditioningtherapy comprises administering an effective amount of fludarabine tothe subject. In preferred embodiments, the conditioning therapycomprises administering an effective amount of a combination ofcyclophosphamide and fludarabine to the subject. Administration of aconditioning therapy prior to CAR T cell therapy may increase theefficacy of the CAR T cell therapy. Methods of conditioning patients forT cell therapy are described in U.S. Pat. No. 9,855,298, which isincorporated herein by reference in its entirety.

In some embodiments, a specific dosage regimen of the present disclosureincludes a lymphodepletion step prior to the administration of themodified T cells. In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide and/or fludarabine.

In some embodiments, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day). In anexemplary embodiment, the dose of cyclophosphamide is about 300mg/m²/day. In some embodiments, the lymphodepletion step includesadministration of fludarabine at a dose of between about 20 mg/m²/dayand about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day,or 60 mg/m²/day). In an exemplary embodiment, the dose of fludarabine isabout 30 mg/m²/day.

In some embodiment, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), andfludarabine at a dose of between about 20 mg/m²/day and about 900mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, ormg/m²/day). In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide at a dose of about 300mg/m²/day, and fludarabine at a dose of about 30 mg/m²/day.

In an exemplary embodiment, the dosing of cyclophosphamide is 300mg/m²/day over three days, and the dosing of fludarabine is 30 mg/m²/dayover three days.

Dosing of lymphodepletion chemotherapy may be scheduled on Days −6 to −4(with a −1 day window, i.e., dosing on Days −7 to −5) relative to T cell(e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

In an exemplary embodiment, for a subject having cancer, the subjectreceives lymphodepleting chemotherapy including 300 mg/m² ofcyclophosphamide by intravenous infusion 3 days prior to administrationof the modified T cells. In an exemplary embodiment, for a subjecthaving cancer, the subject receives lymphodepleting chemotherapyincluding 300 mg/m² of cyclophosphamide by intravenous infusion for 3days prior to administration of the modified T cells.

In an exemplary embodiment, for a subject having cancer, the subjectreceives lymphodepleting chemotherapy including fludarabine at a dose ofbetween about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day,25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplaryembodiment, for a subject having cancer, the subject receiveslymphodepleting chemotherapy including fludarabine at a dose of 30 mg/m²for 3 days.

In an exemplary embodiment, for a subject having cancer, the subjectreceives lymphodepleting chemotherapy including cyclophosphamide at adose of between about 200 mg/m²/day and about 2000 mg/m²/day (e.g., 200mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), and fludarabine at a doseof between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplaryembodiment, for a subject having cancer, the subject receiveslymphodepleting chemotherapy including cyclophosphamide at a dose ofabout 300 mg/m²/day, and fludarabine at a dose of 30 mg/m² for 3 days.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of thecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

It is known in the art that one of the adverse effects followinginfusion of CAR T cells is the onset of immune activation, known ascytokine release syndrome (CRS). CRS is immune activation resulting inelevated inflammatory cytokines. CRS is a known on-target toxicity,development of which likely correlates with efficacy. Clinical andlaboratory measures range from mild CRS (constitutional symptoms and/orgrade-2 organ toxicity) to severe CRS (sCRS; grade ≥3 organ toxicity,aggressive clinical intervention, and/or potentially life threatening).Clinical features include: high fever, malaise, fatigue, myalgia,nausea, anorexia, tachycardia/hypotension, capillary leak, cardiacdysfunction, renal impairment, hepatic failure, and disseminatedintravascular coagulation. Dramatic elevations of cytokines includinginterferon-gamma, granulocyte macrophage colony-stimulating factor,IL-10, and IL-6 have been shown following CAR T-cell infusion. One CRSsignature is elevation of cytokines including IL-6 (severe elevation),IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations inclinically available markers of inflammation including ferritin andC-reactive protein (CRP) have also been observed to correlate with theCRS syndrome. The presence of CRS generally correlates with expansionand progressive immune activation of adoptively transferred cells. Ithas been demonstrated that the degree of CRS severity is dictated bydisease burden at the time of infusion as patients with high tumorburden experience a more sCRS.

Accordingly, the invention provides for, following the diagnosis of CRS,appropriate CRS management strategies to mitigate the physiologicalsymptoms of uncontrolled inflammation without dampening the antitumorefficacy of the engineered cells (e.g., CAR T cells). CRS managementstrategies are known in the art. For example, systemic corticosteroidsmay be administered to rapidly reverse symptoms of sCRS (e.g., grade 3CRS) without compromising initial antitumor response.

In some embodiments, an anti-IL-6R antibody may be administered. Anexample of an anti-IL-6R antibody is the Food and DrugAdministration-approved monoclonal antibody tocilizumab, also known asatlizumab (marketed as Actemra, or RoActemra). Tocilizumab is ahumanized monoclonal antibody against the interleukin-6 receptor(IL-6R). Administration of tocilizumab has demonstrated near-immediatereversal of CRS.

CRS is generally managed based on the severity of the observed syndromeand interventions are tailored as such. CRS management decisions may bebased upon clinical signs and symptoms and response to interventions,not solely on laboratory values alone.

Mild to moderate cases generally are treated with symptom managementwith fluid therapy, non-steroidal anti-inflammatory drug (NSAID) andantihistamines as needed for adequate symptom relief. More severe casesinclude patients with any degree of hemodynamic instability; with anyhemodynamic instability, the administration of tocilizumab isrecommended. The first-line management of CRS may be tocilizumab, insome embodiments, at the labeled dose of 8 mg/kg IV over 60 minutes (notto exceed 800 mg/dose); tocilizumab can be repeated Q8 hours. Ifsuboptimal response to the first dose of tocilizumab, additional dosesof tocilizumab may be considered. Tocilizumab can be administered aloneor in combination with corticosteroid therapy. Patients with continuedor progressive CRS symptoms, inadequate clinical improvement in 12-18hours or poor response to tocilizumab, may be treated with high-dosecorticosteroid therapy, generally hydrocortisone 100 mg IV ormethylprednisolone 1-2 mg/kg. In patients with more severe hemodynamicinstability or more severe respiratory symptoms, patients may beadministered high-dose corticosteroid therapy early in the course of theCRS. CRS management guidance may be based on published standards (Lee etal. (2019) Biol Blood Marrow Transplant,doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev ClinOncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

Features consistent with Macrophage Activation Syndrome (MAS) orHemophagocytic lymphohistiocytosis (HLH) have been observed in patientstreated with CAR-T therapy (Henter, 2007), coincident with clinicalmanifestations of the CRS. MAS appears to be a reaction to immuneactivation that occurs from the CRS, and should therefore be considereda manifestation of CRS. MAS is similar to HLH (also a reaction to immunestimulation). The clinical syndrome of MAS is characterized by highgrade non-remitting fever, cytopenias affecting at least two of threelineages, and hepatosplenomegaly. It is associated with high serumferritin, soluble interleukin-2 receptor, and triglycerides, and adecrease of circulating natural killer (NK) activity.

The modified immune cells comprising CAR of the present invention may beused in a method of treatment as described herein. In one aspect, theinvention includes a method of treating cancer in a subject in needthereof, comprising administering to the subject any one of the modifiedimmune or precursor cells disclosed herein. Yet another aspect of theinvention includes a method of treating cancer in a subject in needthereof, comprising administering to the subject a modified immune orprecursor cell generated by any one of the methods disclosed herein.

One aspect of the invention provides a method of treating prostatecancer in a subject in need thereof, comprising administering to thesubject an effective amount of a modified T cell comprising a chimericantigen receptor (CAR) capable of binding prostate stem cell antigen(PSCA-CAR).

In another aspect, the invention provides a method of treating prostatecancer in a subject in need thereof, comprising administering to thesubject an effective amount of a modified T cell comprising a firstchimeric antigen receptor (CAR) capable of binding prostate stem cellantigen (PSCA-CAR), and a second chimeric antigen receptor (CAR) capableof binding prostate specific membrane antigen (PSMA-CAR), wherein thefirst CAR and the second CAR each comprise an antigen-binding domain, atransmembrane domain, and an intracellular domain.

Another aspect of the invention provides a method of treating prostatecancer in a subject in need thereof, comprising administering to thesubject an effective amount of a modified T cell comprising a bispecificchimeric antigen receptor (CAR) comprising an extracellular domaincomprising an antigen-binding domain capable of binding prostate stemcell antigen (PSCA) and an antigen-binding domain capable of bindingprostate specific membrane antigen (PSMA), a transmembrane domain, andan intracellular domain.

Another aspect of the invention provides a method of treating prostatecancer in a subject in need thereof, comprising administering to thesubject an effective amount of any of the modified T cells contemplatedherein.

Yet another aspect includes a method of treating metastatic castrateresistant prostate cancer in a subject in need thereof, comprisingadministering to the subject an effective amount of a modified T cellcomprising a first chimeric antigen receptor (CAR) capable of bindingprostate stem cell antigen (PSCA-CAR), and a second chimeric antigenreceptor (CAR) capable of binding prostate specific membrane antigen(PSMA-CAR), wherein the first CAR and the second CAR each comprise anantigen-binding domain, a transmembrane domain, and an intracellulardomain.

Also provided is a method of treating metastatic castrate resistantprostate cancer in a subject in need thereof, comprising administeringto the subject an effective amount of a modified T cell comprising abispecific chimeric antigen receptor (CAR) comprising an extracellulardomain comprising an antigen-binding domain capable of binding prostatestem cell antigen (PSCA) and an antigen-binding domain capable ofbinding prostate specific membrane antigen (PSMA), a transmembranedomain, and an intracellular domain.

In another aspect, the invention provides a method of treatingmetastatic castrate resistant prostate cancer in a subject in needthereof, comprising administering to the subject an effective amount ofa modified T cell comprising a chimeric antigen receptor (CAR) capableof binding prostate stem cell antigen (PSCA-CAR), and a dominantnegative receptor.

In another aspect, the invention provides a method of treatingmetastatic castrate resistant prostate cancer in a subject in needthereof, comprising administering to the subject an effective amount ofa modified T cell comprising a chimeric antigen receptor (CAR) capableof binding prostate stem cell antigen (PSCA-CAR), and a switch receptor.

In another aspect, the invention provides a method of treatingmetastatic castrate resistant prostate cancer in a subject in needthereof, comprising administering to the subject an effective amount ofa modified T cell comprising a chimeric antigen receptor (CAR) capableof binding prostate stem cell antigen (PSCA-CAR), and a bispecificantibody, wherein the modified T cell secretes the bispecific antibody.

G. Expansion of Immune Cells

Whether prior to or after modification of cells to express a CAR, thecells can be activated and expanded in number using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example,the T cells of the invention may be expanded by contact with a surfacehaving attached thereto an agent that stimulates a CD3/TCR complexassociated signal and a ligand that stimulates a co-stimulatory moleculeon the surface of the T cells. In particular, T cell populations may bestimulated by contact with an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, T cells can be contacted withan anti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,France) and these can be used in the invention, as can other methods andreagents known in the art (see, e.g., ten Berge et al., Transplant Proc.(1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9):1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2):53-63).

Expanding T cells by the methods disclosed herein can be multiplied byabout 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold,4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000fold, 100,000 fold, 1,000,000 fold, fold, or greater, and any and allwhole or partial integers therebetween. In one embodiment, the T cellsexpand in the range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the chimeric membrane protein.

Another procedure for ex vivo expansion cells is described in U.S. Pat.No. 5,199,942 (incorporated herein by reference). Expansion, such asdescribed in U.S. Pat. No. 5,199,942 can be an alternative or inaddition to other methods of expansion described herein. Briefly, exvivo culture and expansion of T cells comprises the addition to thecellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kitligand. In one embodiment, expanding the T cells comprises culturing theT cells with a factor selected from the group consisting of flt3-L,IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. A cell isolated by the methods disclosed herein can beexpanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold,500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold,3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000fold, fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.In one embodiment, the T cells expand in the range of about 20 fold toabout 50 fold, or more. In one embodiment, human T regulatory cells areexpanded via anti-CD3 antibody coated KT64.86 artificial antigenpresenting cells (aAPCs). Methods for expanding and activating T cellscan be found in U.S. Pat. Nos. 7,754,482, 8,722,400, and 9,555,105,contents of which are incorporated herein in their entirety.

In one embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. Inanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as a transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid, into the expanded population of Tcells, wherein the agent further stimulates the T cell. The agent maystimulate the T cells, such as by stimulating further expansion,effector function, or another T cell function.

H. Methods of Producing Modified Immune Cells

The present disclosure provides methods for producing or generating amodified immune cell or precursor thereof (e.g., a T cell) of theinvention for tumor immunotherapy, e.g., adoptive immunotherapy.

In some embodiments, the CAR is introduced into a cell by an expressionvector. Expression vectors comprising a nucleic acid sequence encoding aCAR of the present invention are provided herein. Suitable expressionvectors include lentivirus vectors, gamma retrovirus vectors, foamyvirus vectors, adeno associated virus (AAV) vectors, adenovirus vectors,engineered hybrid viruses, naked DNA, including but not limited totransposon mediated vectors, such as Sleeping Beauty, Piggybak, andIntegrases such as Phi31. Some other suitable expression vectors includeHerpes simplex virus (HSV) and retrovirus expression vectors.

In certain embodiments, the nucleic acid encoding a CAR is introducedinto the cell via viral transduction. In certain embodiments, the viraltransduction comprises contacting the immune or precursor cell with aviral vector comprising the nucleic acid encoding a CAR. In certainembodiments, the viral vector is an adeno-associated viral (AAV) vector.In certain embodiments, the AAV vector comprises a 5′ ITR and a 3′ITRderived from AAV6. In certain embodiments, the AAV vector comprises aWoodchuck Hepatitis Virus post-transcriptional regulatory element(WPRE). In certain embodiments, the AAV vector comprises apolyadenylation (polyA) sequence. In certain embodiments, the polyAsequence is a bovine growth hormone (BGH) polyA sequence.

Adenovirus expression vectors are based on adenoviruses, which have alow capacity for integration into genomic DNA but a high efficiency fortransfecting host cells. Adenovirus expression vectors containadenovirus sequences sufficient to: (a) support packaging of theexpression vector and (b) to ultimately express the CAR in the hostcell. In some embodiments, the adenovirus genome is a 36 kb, linear,double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acidencoding a CAR) may be inserted to substitute large pieces of adenoviralDNA in order to make the expression vector of the present invention(see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20):1707-1714).

Another expression vector is based on an adeno associated virus (AAV),which takes advantage of the adenovirus coupled systems. This AAVexpression vector has a high frequency of integration into the hostgenome. It can infect nondividing cells, thus making it useful fordelivery of genes into mammalian cells, for example, in tissue culturesor in vivo. The AAV vector has a broad host range for infectivity.Details concerning the generation and use of AAV vectors are describedin U.S. Pat. Nos. 5,139,941 and 4,797,368.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retroviral vector is constructed by inserting a nucleicacid (e.g., a nucleic acid encoding a CAR) into the viral genome atcertain locations to produce a virus that is replication defective.Though the retroviral vectors are able to infect a broad variety of celltypes, integration and stable expression of the CAR requires thedivision of host cells.

Lentiviral vectors are derived from lentiviruses, which are complexretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2)and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors havebeen generated by multiply attenuating the HIV virulence genes, forexample, the genes env, vif, vpr, vpu and nef are deleted making thevector biologically safe. Lentiviral vectors are capable of infectingnon-dividing cells and can be used for both in vivo and ex vivo genetransfer and expression, e.g., of a nucleic acid encoding a CAR (see,e.g., U.S. Pat. No. 5,994,136).

Expression vectors including a nucleic acid of the present disclosurecan be introduced into a host cell by any means known to persons skilledin the art. The expression vectors may include viral sequences fortransfection, if desired. Alternatively, the expression vectors may beintroduced by fusion, electroporation, biolistics, transfection,lipofection, or the like. The host cell may be grown and expanded inculture before introduction of the expression vectors, followed by theappropriate treatment for introduction and integration of the vectors.The host cells are then expanded and may be screened by virtue of amarker present in the vectors. Various markers that may be used areknown in the art, and may include hprt, neomycin resistance, thymidinekinase, hygromycin resistance, etc. As used herein, the terms “cell,”“cell line,” and “cell culture” may be used interchangeably. In someembodiments, the host cell an immune cell or precursor thereof, e.g., aT cell, an NK cell, or an NKT cell.

The present invention also provides genetically engineered cells whichinclude and stably express a CAR of the present disclosure. In someembodiments, the genetically engineered cells are genetically engineeredT-lymphocytes (T cells), naive T cells (TN), memory T cells (forexample, central memory T cells (TCM), effector memory cells (TEM)),natural killer cells (NK cells), and macrophages capable of giving riseto therapeutically relevant progeny. In certain embodiments, thegenetically engineered cells are autologous cells. In certainembodiments, the modified cell is resistant to T cell exhaustion.

Modified cells (e.g., comprising a CAR) may be produced by stablytransfecting host cells with an expression vector including a nucleicacid of the present disclosure. Additional methods for generating amodified cell of the present disclosure include, without limitation,chemical transformation methods (e.g., using calcium phosphate,dendrimers, liposomes and/or cationic polymers), non-chemicaltransformation methods (e.g., electroporation, optical transformation,gene electrotransfer and/or hydrodynamic delivery) and/or particle-basedmethods (e.g., impalefection, using a gene gun and/or magnetofection).Transfected cells expressing a CAR of the present disclosure may beexpanded ex vivo.

Physical methods for introducing an expression vector into host cellsinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells including vectors and/or exogenous nucleic acids arewell-known in the art. See, e.g., Sambrook et al. (2001), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.Chemical methods for introducing an expression vector into a host cellinclude colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K &K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,AL). Stock solutions of lipids in chloroform or chloroform/methanol canbe stored at about −20° C. Chloroform may be used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). Compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, molecular biology assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;biochemistry assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, the nucleic acids introduced into the host cell areRNA. In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA may be produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA may be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA.

PCR may be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary,” as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary. Substantiallycomplementary sequences are able to anneal or hybridize with theintended DNA target under annealing conditions used for PCR. The primerscan be designed to be substantially complementary to any portion of theDNA template. For example, the primers can be designed to amplify theportion of a gene that is normally transcribed in cells (the openreading frame), including 5′ and 3′ UTRs. The primers may also bedesigned to amplify a portion of a gene that encodes a particular domainof interest. In one embodiment, the primers are designed to amplify thecoding region of a human cDNA, including all or portions of the 5′ and3′ UTRs. Primers useful for PCR are generated by synthetic methods thatare well known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA. 5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

In some embodiments, the RNA is electroporated into the cells, such asin vitro transcribed RNA. Any solutes suitable for cell electroporation,which can contain factors facilitating cellular permeability andviability such as sugars, peptides, lipids, proteins, antioxidants, andsurfactants can be included.

In some embodiments, a nucleic acid encoding a CAR of the presentdisclosure will be RNA, e.g., in vitro synthesized RNA. Methods for invitro synthesis of RNA are known in the art; any known method can beused to synthesize RNA comprising a sequence encoding a CAR. Methods forintroducing RNA into a host cell are known in the art. See, e.g., Zhaoet al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising anucleotide sequence encoding a CAR into a host cell can be carried outin vitro, ex vivo or in vivo. For example, a host cell (e.g., an NKcell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro orex vivo with RNA comprising a nucleotide sequence encoding a CAR.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

In another aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

I. Pharmaceutical Compositions and Formulations

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of any of themodified cells contemplated herein. Also provided are populations ofimmune cells of the invention, compositions containing such cells and/orenriched for such cells, such as in which cells expressing the CAR makeup at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more of the total cells in the composition or cells of acertain type such as T cells or CD8+ or CD4+ cells. Among thecompositions are pharmaceutical compositions and formulations foradministration, such as for adoptive cell therapy. Also provided aretherapeutic methods for administering the cells and compositions tosubjects, e.g., patients.

Also provided are compositions including the cells for administration,including pharmaceutical compositions and formulations, such as unitdose form compositions including the number of cells for administrationin a given dose or fraction thereof. The pharmaceutical compositions andformulations generally include one or more optional pharmaceuticallyacceptable carrier or excipient. In some embodiments, the compositionincludes at least one additional therapeutic agent.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. A “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical formulation, otherthan an active ingredient, which is nontoxic to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative. In some aspects, thechoice of carrier is determined in part by the particular cell and/or bythe method of administration. Accordingly, there are a variety ofsuitable formulations. For example, the pharmaceutical composition cancontain preservatives. Suitable preservatives may include, for example,methylparaben, propylparaben, sodium benzoate, and benzalkoniumchloride. In some aspects, a mixture of two or more preservatives isused. The preservative or mixtures thereof are typically present in anamount of about 0.0001% to about 2% by weight of the total composition.Carriers are described, e.g., by Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecells, preferably those with activities complementary to the cells,where the respective activities do not adversely affect one another.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended. Thus, in some embodiments,the pharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and/or vincristine. Thepharmaceutical composition in some embodiments contains the cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. The desired dosage can bedelivered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, the cellpopulations are administered parenterally. The term “parenteral,” asused herein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecells are administered to the subject using peripheral systemic deliveryby intravenous, intraperitoneal, or subcutaneous injection. Compositionsin some embodiments are provided as sterile liquid preparations, e.g.,isotonic aqueous solutions, suspensions, emulsions, dispersions, orviscous compositions, which may in some aspects be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyoi (for example, glycerol, propylene glycol, liquid polyethyleneglycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can contain auxiliary substancessuch as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. It will be readily apparent to those skilled in the art thatother suitable modifications and adaptations of the methods describedherein may be made using suitable equivalents without departing from thescope of the embodiments disclosed herein. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the present invention. All such modifications areintended to be within the scope of the claims appended hereto. Havingnow described certain embodiments in detail, the same will be moreclearly understood by reference to the following examples, which areincluded for purposes of illustration only and are not intended to belimiting.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods

Cell lines and primary human T lymphocyte cultures: Primary human CD4and CD8 T cells were isolated from healthy volunteer donors followingleukapheresis by negative selection using RosetteSep Kits (Stem CellTechnologies). All specimens were collected under a UniversityInstitutional Review Board-approved protocol, and written informedconsent was obtained from each donor. Mixed primary human CD4 and CD8 Tcells (1:1) were stimulated with anti-CD3/CD28 Dynabeads (LifeTechnologies).

CAR constructs and lentiviral transduction: A PSCA CAR comprised of anscFv from a humanized anti-PSCA Ab (2B3) was constructed and cloned intoretroviral vector MSGV. The scFv domain against PSCA was synthesizedand/or amplified by PCR, linked to CD8 transmembrane domain and 4-1BB,CD28, ICOS or ICOS.YMNM, and CD3 zeta intracellular signaling domains,and subcloned into pTRPE lentiviral vectors. T cells were transducedwith lentiviral vectors at an MOI of 5.

Flow cytometry: Flow cytometry was used to determine the transductionefficiency of transduced cells following staining with biotin-labeledpolyclonal anti-mouse F(ab)2 antibody (Jackson Immunoresearch). Thefollowing antibodies were used in flow cytometry experiments: PEconjugated Streptavidin. Data acquisition was performed on FACSCalibur(BC Biosciences) and analyzed via FlowJo.

CD107a assay: Cells were plated at an E:T of 1:2 (1×105 effectors:2×105) in 160 of R10 medium in a 96-well plate. Of note, 20 μL ofphycoerythrin-labeled anti-CD107a Ab was added and the plate wasincubated at 37° C. for 1 hour before adding Golgi Stop (2 mL Golgi Stopin 3 mL R10 medium, 20 mL/well; BD Biosciences, 51-2092 KZ) andincubating for another 2.5 hours. Then 5 mL FITC-anti-CD8 and 5 mLstreptavidin-allophycocyanin (APC)-anti-CD3 were added and incubated at37° C. for 30 minutes. After incubation, the samples were washed withFACS buffer and analyzed by flow cytometry.

Enzyme-linked immunosorbent assay (ELISA): Target cells were washed andsuspended at 1×10⁶ cells/mL in R10 medium. Of note, 100 μL each targetcell type were added in triplicate to a 96-well round bottom plate(Corning). Effector T cells were washed and resuspended at 1×10⁶cells/mL in R10 cells and then 100 μL of T cells were combined withtarget cells in the indicated wells. The plates were incubated at 37° C.for 18 to 24 hours. After the incubation, supernatant was harvested andsubjected to an ELISA assay (eBioscience).

Luciferase-based cytolytic T-cell (CTL) assay: Briefly, Click beetlegreen luciferase (CBG)-T2A-eGFP was lenti-virally transduced into PSCAtumor cells and sorted for GFP expression. Tumor cells were incubatedwith different ratios of T cells 8 hours at 37° C. Of note, 100 mL ofthe mixture was transferred to a 96-well white luminometer plate, 100 mLof substrate was added, and the luminescence was immediately determined.Results are reported as percent killing based on luciferase activity inwells with tumor, but no T cells. (% killing=100−((RLU from well witheffector and target cell coculture)/(RLU from well with targetcells)×100).

Example 1

Various prostate stem cell antigen (PSCA) specific CARs were generatedherein. The antigen binding domain was derived from a humanizedanti-PSCA antibody (2B3) (U.S. Patent Publication No. US2010/0297004,the contents of which is hereby incorporated by reference in itsentirety) (FIG. 1 ). The 2B3 scFv was used in combination with variousintracellular domains including 4-1BB and CD3 zeta (2B3.BBZ CAR), CD28and CD3 zeta (2B3.28Z CAR), ICOS and CD3 zeta (2B3.ICOSZ CAR), and amutated ICOS (ICOS.YMNM CAR) and CD3 zeta (2B3.ICOS.YMNM CAR). PSCA CARscomprising PD1-CD28 switch receptors, TGFbR/IL12R switch receptors, anda dominant negative receptor (TGFbRDN) were also generated. Dual CARscomprising specificity for PSCA and PSMA were also developed. PSCA CARswere also used in combination with bispecific antibodies (e.g.aPD-L1/CD28 or aTGFbRII-CD28).

CAR expression was measured in T cells co-electroporated with in vitrotranscribed RNA of a PSCA CAR (2B3.BBZ) and a PMSA CAR (J591.BBZ) (FIG.2 upper panel). PD-L1-Fc staining of a PSCA CAR co-electroporated with abispecific antibody 10A5-1412 (an aPDL1-aCD28 bispecific Ab) orTGFB3-1412 (an aTGFbRII-aCD28 bispecific Ab) is shown in FIG. 2 , lowerpanel.

CD107a was measured in RNA co-electroporated T cells stimulated withPC3-PSCA-PSMA or K562 (FIG. 3 ).

T cells lentivirally transduced with a PSCA (2B3) CAR with mutated ICOSsignaling domain (ICOS.YMNM) showed improved lytic capability, decreasedcytokine production in vitro and equivalent in vivo antitumor activitiesas a 4-1BB signaling PSCA CAR (FIG. 4 and FIG. 6 ). PSCA CARs witheither ICOS or ICOS.YMNM signaling domain were constructed and clonedinto a lentiviral vector. The CAR expression levels were comparable witheither 4-1BB or CD28 signaling domain CARs (FIG. 4 upper). Followingstimulation with PSCA positive cell lines PC3.PSCA.PMSA.CBG orPC3.PSCA.PMSA.CBG.PD-L1, cytokine production (IFN-gamma) was measured(FIG. 4 lower panel).

Results from a CD107a assay for T cells expressing PSCA CARs with eitherICOS or ICOS.YMNM signaling domain are shown in FIG. 5A. FIG. 5Billustrates results from a killing assay for T cells expressing PSCACARs with either ICOS or ICOS.YMNM signaling domain against tumor linePC3-PSCA.

FIG. 6 illustrates BLI results from a PC3-PSCA-CBG-PDL1 tumor model. Forlentivirally transduced (LVV TD) T cells, 1e⁶ cells were transduced permouse, intravenously (i.v.) at day 21 post tumor inoculation. BLI (theaverage radiace) of tumors (FIG. 7 ) and tumor sizes (FIG. 8 ) weremeasured after subcutaneous injection with various PSCA CARs.

Bi-specific CARs were also generated herein. Vectors used for studiesencoding the PSMA (2F5 scFv) and PSCA-targeted CARs, linked with aGly4Ser element are depicted in FIG. 9A. Surface expression of the CARson lentivirus transduced CAR T cells at the end of the primary expansionis depicted in FIG. 9B. The percentage of lentivirus transduced CAR Tcells that express the PSMA or PSCA-CAR or bispecific CAR was measuredby staining with human recombinant PSMA-Fc and PSCA-His protein andanalyzed by flow cytometry (FIG. 9C). CAR T cells expressing theindicated scFv were co-cultured with targets for 4 hours and thepercentage CD107a expression was quantified on CD8 positive cells (FIG.9D).

Bi-specific CAR T cells were co-cultured with PC3-PSCA cells(Effect:Target=1:1). Supernatants were obtained 24 hours afterco-culture, and cytokine production was analyzed by ELISA (FIG. 10A).Bi-specific CAR T cells were tested for their cytolytic activity atvarious E:T ratios for 8 hours against PC3-PSCA cells (FIG. 10B).Bi-specific CAR T cells were co-cultured with PC3-PSMA cells(Effect:Target=1:1). Supernatants were obtained 24 hours afterco-culture, and cytokine production was analyzed by ELISA (FIG. 10C).Bi-specific CAR T cells were tested for their cytolytic activity atvarious E:T ratios for 8 hours against PC3-PSMA cells (FIG. 10D).

FIGS. 11A-11B illustrate the finding that TGFbR-IL12R switch receptorscan boost T cell function. FIG. 11A, upper right panel, shows IFN-gammaproduction of NK cells trasferred with TGFbR-IL12R co-cultured withK562, with or without TGFb1 in the cultures. FIG. 11A, lower rightpanel, shows pSmad staining of T cells transferred with TGFbR-IL12Rswitch receptors after stimulation with TGF beta. FIG. 11B showscytokine production of NY-ESO-1 positive tumors stimulated NY-ESO-1 TCRtransduced T cells, co-transferred with TGFbR-IL12R switch receptors.

OTHER EMBODIMENTS

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-4. (canceled)
 5. A nucleic acid comprising a polynucleotide sequenceencoding a CAR comprising an antigen binding domain capable of bindingprostate stem cell antigen (PSCA), a transmembrane domain, and anintracellular domain; wherein the antigen binding domain comprises: (a)a heavy chain variable region that comprises three heavy chaincomplementarity determining regions (HCDRs), wherein HCDR1 comprises theamino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprises the amino acidsequence WIDPENGDTEFVPKF QG (SEQ ID NO: 2), and HCDR3 comprises theamino acid sequence TGGF (SEQ ID NO: 3); and (b) a light chain variableregion that comprises three light chain complementarity determiningregions (LCDRs), wherein LCDR1 comprises the amino acid sequenceSASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acid sequenceDTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequenceQQWSSSPFT (SEQ ID NO: 6).
 6. The nucleic acid of claim 5, wherein theCAR comprises a polynucleotide sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20, 22, 24, or 26.7-8. (canceled)
 9. A vector comprising the nucleic acid of claim
 6. 10.(canceled)
 11. The vector of claim 9, wherein the vector is anexpression vector. 12-16. (canceled)
 17. A method of treating a diseasein a subject in need thereof, comprising administering to the subject aneffective amount of a modified cell comprising a chimeric antigenreceptor (CAR), wherein the CAR comprises an antigen binding domaincapable of binding prostate stem cell antigen (PSCA), a transmembranedomain, and an intracellular domain; and wherein the antigen bindingdomain comprises: (a) a heavy chain variable region that comprises threeheavy chain complementarity determining regions (HCDRs), wherein HCDR1comprises the amino acid sequence DYYIH (SEQ ID NO: 1), HCDR2 comprisesthe amino acid sequence WIDPENGDTEFVPKF QG (SEQ ID NO: 2), and HCDR3comprises the amino acid sequence TGGF (SEQ ID NO: 3); and (b) a lightchain variable region that comprises three light chain complementaritydetermining regions (LCDRs), wherein LCDR1 comprises the amino acidsequence SASSSVRFIHW (SEQ ID NO: 4), LCDR2 comprises the amino acidsequence DTSKLAS (SEQ ID NO: 5), and LCDR3 comprises the amino acidsequence QQWSSSPFT (SEQ ID NO: 6).
 18. The method of claim 17, whereinthe disease is a cancer.
 19. The method of claim 18, wherein the diseaseis prostate cancer.
 20. The method of claim 18, wherein the subject is ahuman.
 21. The method of claim 17, wherein the CAR comprises: (a) theheavy chain variable region comprising an amino acid sequence at least90% identical to SEQ ID NO: 7; and/or (b) the light chain variableregion comprising an amino acid sequence at least 90%, identical to SEQID NO:
 8. 22. The method of claim 17, wherein the antigen binding domainis a single-chain variable fragment (scFv) and comprises an amino acidsequence at least 90% identical to SEQ ID NO:
 9. 23. The method of claim17, wherein the intracellular domain comprises: (a) a costimulatorydomain of a protein selected from the group consisting of proteins inthe TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7,LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I,TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variantthereof, or an intracellular domain derived from a killerimmunoglobulin-like receptor (KIR); and/or (b) CD3 zeta intracellulardomain.
 24. The method of claim 17, wherein the CAR comprises the aminoacid sequence of SEQ ID NO: 21, 23, 25, or
 27. 25. The method of claim17, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 21.26. The method of claim 17, wherein the modified immune cell furthercomprises a CAR capable of binding prostate specific membrane antigen(PSMA), and wherein the PSMA-CAR comprises an antigen-binding domain, atransmembrane domain, and an intracellular domain, and the antigenbinding domain of the PSMA-CAR comprises: (a) a heavy chaincomplementarity determining region (HCDR) 1 comprising the amino acidsequence of SEQ ID NO: 28, HCDR 2 comprising the amino acid sequence ofSEQ ID NO: 29, and HCDR 3 comprising the amino acid sequence of SEQ IDNO: 30; and a light chain complementarity determining region (LCDR) 1comprising the amino acid sequence of SEQ ID NO: 31, LCDR 2 comprisingthe amino acid sequence of SEQ ID NO: 32, and LCDR 3 comprising theamino acid sequence of SEQ ID NO: 33; or (b) a heavy chaincomplementarity determining region (HCDR) 1 comprising the amino acidsequence of SEQ ID NO: 68, HCDR 2 comprising the amino acid sequence ofSEQ ID NO: 69, and HCDR 3 comprising the amino acid sequence of SEQ IDNO: 70; and a light chain complementarity determining region (LCDR) 1comprising the amino acid sequence of SEQ ID NO: 71, LCDR 2 comprisingthe amino acid sequence of SEQ ID NO: 72, and LCDR 3 comprising theamino acid sequence of SEQ ID NO:
 73. 27. The method of claim 26,wherein the PSMA CAR comprises an amino acid sequence at least about 90%identical to SEQ ID NO: 67, 87, 89, 91, 93, 95, or
 97. 28. The method ofclaim 26, wherein: (a) the PSCA CAR comprises the amino acid sequence ofSEQ ID NO: 21 and the PSMA CAR comprises an amino acid sequence at leastabout about 90% identical to SEQ ID NO: 67; or (b) the PSCA CARcomprises the amino acid sequence of SEQ ID NO: 21; and the PSMA CARcomprises an amino acid sequence at least about 90%, identical to SEQ IDNO:
 87. 29. The method of claim 26, wherein the intracellular domain ofthe PSCA CAR and the PSMA CAR each comprises: (a) a 4-1BB intracellulardomain; (b) a CD28 intracellular domain; (c) an ICOS intracellulardomain; or (d) an ICOS(YMNM).
 30. The method of claim 26, wherein thecostimulatory domain of the PSCA CAR is different from the costimulatorydomain of the PSMA CAR.
 31. The method of claim 26, wherein the modifiedcell comprises the amino acid sequence of SEQ ID NO: 40, 42, 80, 81, or82.
 32. A method of treating a disease in a subject in need thereof,comprising administering to the subject an effective amount of amodified cell comprising a vector comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprise anantigen binding domain capable of binding prostate stem cell antigen(PSCA), a transmembrane domain, and an intracellular domain, and whereinthe vector comprises a nucleic acid sequence at least 85% identical toSEQ ID NO: 43 and a nucleic acid sequence at least 85% identical to SEQID NO:
 44. 33. The method of claim 32, wherein the vector comprises anucleic acid sequence at least 85% identical to SEQ ID NO: 20, 22, 24,or
 26. 34. The method of claim 32, wherein the vector further comprises:(a) a nucleic acid sequence at least 85% identical to SEQ ID NO: 46 anda nucleic acid sequence at least 85% identical to SEQ ID NO: 47; or (b)a nucleic acid sequence at least about 90% identical to SEQ ID NO: 88,90, 92, 94, 96, or
 98. 35. The method of claim 32, wherein the vectorcomprises the nucleic acid sequence of SEQ ID NO: 39, 41, or
 79. 36. Themethod of claim 31, wherein: (a) the disease is a cancer; (b) thedisease is prostate cancer; or (c) the subject is a human.